Improvement of Rice Biomass Yield through QTL-Based Selection

Biomass yield of rice (Oryza sativa L.) is an important breeding target, yet it is not easy to improve because the trait is complex and phenotyping is laborious. Using progeny derived from a cross between two high-yielding Japanese cultivars, we evaluated whether quantitative trait locus (QTL)-based selection can improve biomass yield. As a measure of biomass yield, we used plant weight (aboveground parts only), which included grain weight and stem and leaf weight. We measured these and related traits in recombinant inbred lines. Phenotypic values for these traits showed a continuous distribution with transgressive segregation, suggesting that selection can affect plant weight in the progeny. Four significant QTLs were mapped for plant weight, three for grain weight, and five for stem and leaf weight (at α = 0.05); some of them overlapped. Multiple regression analysis showed that about 43% of the phenotypic variance of plant weight was significantly explained (P < 0.0001) by six of the QTLs. From F₂ plants derived from the same parental cross as the recombinant inbred lines, we divergently selected lines that carried alleles with positive or negative additive effects at these QTLs, and performed successive selfing. In the resulting F₆ lines and parents, plant weight significantly differed among the genotypes (at α = 0.05). These results demonstrate that QTL-based selection is effective in improving rice biomass yield.     

Quantitative trait loci mapping of adult-plant resistance to powdery mildew in Chinese wheat cultivar Lumai 21

Powdery mildew, caused by Blumeria graminis f. sp. tritici, is a major fungal disease in common wheat (Triticum aestivum L.) worldwide. The Chinese winter wheat cultivar Lumai 21 has shown good and stable adult plant resistance for 19 years. The aim of this study was to map quantitative trait loci (QTLs) for resistance to powdery mildew in a population of 200 F₃ lines from the cross Lumai 21/Jingshuang 16. The population was tested for powdery mildew reaction in Beijing and Anyang in the 2005–2006 and 2006–2007 cropping seasons, providing data for 4 environments. A total of 1,375 simple sequence repeat (SSR) markers were screened for associations with powdery mildew reactions, initially in bulked segregant analysis. Based on the mean disease values averaged across environments, broad-sense heritabilities of maximum disease severity and area under the disease progress curve were 0.96 and 0.77, respectively. Three QTLs for adult plant resistance were detected by inclusive composite interval mapping. These were designated QPm.caas-2BS, QPm.caas-2BL and QPm.caas-2DL, respectively, and explained from 5.4 to 20.6% of the phenotypic variance across environments. QPm.caas-2BS and QPm.caas-2DL were likely new adult plant resistance QTLs flanked by SSR markers Xbarc98–Xbarc1147 and Xwmc18–Xcfd233, respectively. These markers could be useful for improving wheat powdery mildew resistance in breeding programs.     

Effects of Induced Resistance on Disease Severity/Yield Relations in Mildewed Barley

Field experiments showed that the treatment of winter barley with microbial metabolites produced by a Bacillus subtilis strain led to increased yields in spite of a remaining mildew infection. Disease severity/yield relations obtained on a single tiller basis for either mildew infection at EC 75 or area under disease progress curve (AUDPC) were negatively correlated for untreated plants (R²= 89%, 94%) while this relation did not exist for inducer-treated ones (R²= 10%, 13%). Despite an increasing infection density. yields of main tillers of inducertreated plants were not decreased. On the other hand area under green leaf area curve (AUGLAC) showed a higher correlation with grain yield (R²= 89%) of inducer-treated plants. Possible explanations for the mitigated damaging effect of powdery mildew were expected in carbohydrate metabolism. especially carbohydrate formation and translocation. Assimilation rates of flag leaves of inducer-treated barley with similar infection densities to those of untreated plants were increased over a prolonged period and even exceeded those of non infected ones. In inducer-treated plants the export of ¹⁴CO₂ from flag leaves into ears remained unimpaired by mildew infection and the allocation of assimilates to grains was highest at late stages of grain filling. Obviously plants were stimulated by inducer treatments to compensate for the damaging effect of powdery mildew and to tolerate mildew infection without yield loss.     

Water deficits and reproduction in maize : response of the reproductive tissue to water deficits at anthesis and mid-grain fill

Reproductive development in maize (Zea mays L.) is vulnerable to plant water deficits during anthesis but becomes less sensitive as reproduction progresses. To determine whether changes in tissue water status correlated with the change in sensitivity, we examined the water potential (Ψ_w), osmotic potential (Ψ_s), and turgor of reproductive tissues during a short-term water deficit imposed at anthesis or mid-grain fill. Plants were grown in controlled environments in soil. At anthesis, leaf, husk, silk, and ovary Ψ_w of control plants was similar (−0.5 to −0.65 megapascal) at midday. When water was withheld, Ψ_w decreased to −1.75, −1.3, −1.2, and −1.0 megapascal in these tissues. Net water uptake by the ovaries was inhibited, but final dry weight, solute content, and total extractable carbohydrates were similar to the controls. At mid-grain fill, leaf, husk, grain, and embryo Ψ_w of control plants were −0.55, −0.35, −0.75, and −0.80 megapascal at midday. When water was withheld, leaf and husk Ψ_w decreased to −2.4 and −1.4 megapascal within 6 days. However, grain and embryo Ψ_w remained within 0.15 megapascal of control values. The grain continued to accumulate dry matter despite a net loss of water and a reduction in total solute content. These results indicate that the response of the reproductive tissues to plant water deficits varies with stage of grain development. The maintenance of a favorable water status only after grain filling is under way may explain, at least in part, the high sensitivity to plant water deficits early in reproductive development and the decrease in sensitivity as reproduction progresses.     

Molecular Interactions of Alzheimer's Biomarker FDDNP with Aβ Peptide

All-atom explicit solvent model and replica exchange molecular dynamics were used to investigate binding of Alzheimer''s biomarker FDDNP to the Aβ_10–40 monomer. At low and high concentrations, FDDNP binds with high affinity to two sites in the Aβ_10–40 monomer located near the central hydrophobic cluster and in the C-terminal. Analysis of ligand- Aβ_10–40 interactions at both concentrations identifies hydrophobic effect as a main binding factor. However, with the increase in ligand concentration the interactions between FDDNP molecules also become important due to strong FDDNP self-aggregation propensity and few specific binding locations. As a result, FDDNP ligands partially penetrate the core of the Aβ_10–40 monomer, forming large self-aggregated clusters. Ligand self-aggregation does not affect hydrophobic interactions as a main binding factor or the location of binding sites in Aβ_10–40. Using the Aβ_10–40 conformational ensemble in ligand-free water as reference, we show that FDDNP induces minor changes in the Aβ_10–40 secondary structure at two ligand concentrations studied. At the same time, FDDNP significantly alters the peptide tertiary fold in a concentration-dependent manner by redistributing long-range, side-chain interactions. We argue that because FDDNP does not change Aβ_10–40 secondary structure, its antiaggregation effect is likely to be weak. Our study raises the possibility that FDDNP may serve as a biomarker of not only Aβ fibril species, but of monomers as well.     

Pm50: a new powdery mildew resistance gene in common wheat derived from cultivated emmer

Fungal diseases of wheat, including powdery mildew, cause significant crop, yield and quality losses throughout the world. Knowledge of the genetic basis of powdery mildew resistance will greatly support future efforts to develop and cultivate resistant cultivars. Studies were conducted on cultivated emmer-derived wheat line K2 to identify genes involved in powdery mildew resistance at the seedling and adult plant growth stages using a BC₁ doubled haploid population derived from a cross between K2 and susceptible cultivar Audace. A single gene was located distal to microsatellite marker Xgwm294 on the long arm of chromosome 2A. Quantitative trait loci (QTL) analysis indicated that the gene was also effective at the adult plant stage, explaining up to 79.0 % of the variation in the progeny. Comparison of genetic maps indicated that the resistance gene in K2 was different from Pm4, the only other formally named resistance gene located on chromosome 2AL, and PmHNK54, a gene derived from Chinese germplasm. The new gene was designated Pm50.     

Hypersensitive cell death and papilla formation in barley attacked by the powdery mildew fungus are associated with hydrogen peroxide but not with salicylic acid accumulation

We analyzed the pathogenesis-related generation of H₂O₂ using the microscopic detection of 3,3-diaminobenzidine polymerization in near-isogenic barley (Hordeum vulgare L.) lines carrying different powdery mildew (Blumeria graminis f.sp. hordei) resistance genes, and in a line expressing chemically activated resistance after treatment with 2,6-dichloroisonicotinic acid (DCINA). Hypersensitive cell death in Mla12 and Mlg genotypes or after chemical activation by DCINA was associated with H₂O₂ accumulation throughout attacked cells. Formation of cell wall appositions (papillae) mediated in Mlg and mlo5 genotypes and in DCINA-activated plants was paralleled by H₂O₂ accumulation in effective papillae and in cytosolic vesicles of up to 2 μm in diameter near the papillae. H₂O₂ was not detected in ineffective papillae of cells that had been successfully penetrated by the fungus. These findings support the hypothesis that H₂O₂ may play a substantial role in plant defense against the powdery mildew fungus. We did not detect any accumulation of salicylic acid in primary leaves after inoculation of the different barley genotypes, indicating that these defense responses neither relied on nor provoked salicylic acid accumulation in barley.     

Molecular detection of a gene effective against powdery mildew in the wheat cultivar Liangxing 66

Since it was commercialized in 2008, Liangxing 66 is one of the most widely grown cultivars of wheat (Triticum aestivum L.) in winter and facultative wheat-producing regions in northern China. This cultivar displays broad-spectrum resistance to isolates of powdery mildew. To identify the powdery mildew resistance gene in Liangxing 66, genetic analysis and molecular mapping were conducted using the F₂ populations and F_2:3 families derived from the reciprocal crosses of Liangxing 66 and the susceptible cultivar Jingshuang 16. A single dominant gene, tentatively designated PmLX66, conferred resistance in Liangxing 66 to the powdery mildew isolate E09. The results of molecular mapping indicated that this gene was located on the short arm of chromosome 5D and flanked by SCAR203 and Xcfd81 at genetic distances of 0.4 and 2.8?cM, respectively, which is similar to the position of locus Pm2. However, PmLX66 and Pm2 showed different reactions to five of the 42 isolates of powdery mildew tested. Together, these results indicated that PmLX66 was most likely an allele of Pm2. Based on its superior yield and agronomic performance, in combination with powdery mildew resistance, Liangxing 66 is useful as a promising parent for control of powdery mildew and for the development of new disease-resistant cultivars.     

Molecular Plasticity Regulates Oligomerization and Cytotoxicity of the Multipeptide-length Amyloid-β Peptide Pool

Current therapeutic approaches under development for Alzheimer disease, including γ-secretase modulating therapy, aim at increasing the production of Aβ_1–38 and Aβ_1–40 at the cost of longer Aβ peptides. Here, we consider the aggregation of Aβ_1–38 and Aβ_1–43 in addition to Aβ_1–40 and Aβ_1–42, in particular their behavior in mixtures representing the complex in vivo Aβ pool. We demonstrate that Aβ_1–38 and Aβ_1–43 aggregate similar to Aβ_1–40 and Aβ_1–42, respectively, but display a variation in the kinetics of assembly and toxicity due to differences in short timescale conformational plasticity. In biologically relevant mixtures of Aβ, Aβ_1–38 and Aβ_1–43 significantly affect the behaviors of Aβ_1–40 and Aβ_1–42. The short timescale conformational flexibility of Aβ_1–38 is suggested to be responsible for enhancing toxicity of Aβ_1–40 while exerting a cyto-protective effect on Aβ_1–42. Our results indicate that the complex in vivo Aβ peptide array and variations thereof is critical in Alzheimer disease, which can influence the selection of current and new therapeutic strategies.     

RAPD analysis of local bell pepper genotypes in relation with powdery mildew incidence and fruit yield: Managing disease by single chemical molecule

Abstract

A field study was conducted to investigate powdery mildew disease incidence and fruit yield of locally available bell pepper genotypes by Kharif 2004 and Rabi 2005. The study includes a single chemical molecule in managing the powdery mildew disease during both the seasons. Field studies include five chemical treatments (T₁ to T₅) plus one water spray control and an untreated control in five replications each. The result shows that out all treatments T₅ was best in controlling the powdery mildew disease in genotype Indra by 77.1 and 73.7% of disease decrease over control and 85.3 and 95.7% increase in yield over control respectively during Kharif and Rabi. The next best performing genotypes were Arka Basant and California Wonder in terms of less disease incidence and high yield. Further, the study was extended to relate the selected varieties for its genetic diversity using Random Amplified Polymorphic DNAs, with respect to the disease incidence and yield. The highest genetic dissimilarity of 49% was noticed between the genotypes Indra and Arka Gaurav having contrasting characters such as high and low fruit yield, respectively. The lowest genetic diversity of 21% was noticed between the genotypes Arka Mohini and California Wonder that were similar in most of the morphological characters, fruit yield and susceptibility to the powdery mildew disease. Based on the molecular data, a remarkable difference was exhibited by the commercially grown bell pepper genotypes.     

Transcript-Based Cloning of RRP46, a Regulator of rRNA Processing and R Gene–Independent Cell Death in Barley–Powdery Mildew Interactions

Programmed cell death (PCD) plays a pivotal role in plant development and defense. To investigate the interaction between PCD and R gene–mediated defense, we used the 22K Barley1 GeneChip to compare and contrast time-course expression profiles of Blumeria graminis f. sp hordei (Bgh) challenged barley (Hordeum vulgare) cultivar C.I. 16151 (harboring the Mla6 powdery mildew resistance allele) and its fast neutron–derived Bgh-induced tip cell death1 mutant, bcd1. Mixed linear model analysis identified genes associated with the cell death phenotype as opposed to R gene–mediated resistance. One-hundred fifty genes were found at the threshold P value < 0.0001 and a false discovery rate <0.6%. Of these, 124 were constitutively overexpressed in the bcd1 mutant. Gene Ontology and rice (Oryza sativa) alignment-based annotation indicated that 68 of the 124 overexpressed genes encode ribosomal proteins. A deletion harboring six genes on chromosome 5H cosegregates with bcd1-specified cell death and is associated with misprocessing of rRNAs but segregates independent of R gene–mediated resistance. Barley stripe mosaic virus-induced gene silencing of one of the six deleted genes, RRP46 (rRNA-processing protein 46), phenocopied bcd1-mediated tip cell death. These findings suggest that RRP46, a critical component of the exosome core, mediates RNA processing and degradation involved in cell death initiation as a result of attempted penetration by Bgh during the barley–powdery mildew interaction but is independent of gene-for-gene resistance.     

Transient transformation of Podosphaera xanthii by electroporation of conidia

Background

Powdery mildew diseases are a major phytosanitary issue causing important yield and economic losses in agronomic, horticultural and ornamental crops. Powdery mildew fungi are obligate biotrophic parasites unable to grow on culture media, a fact that has significantly limited their genetic manipulation. In this work, we report a protocol based on the electroporation of fungal conidia, for the transient transformation of Podosphaera fusca (synonym Podosphaera xanthii), the main causal agent of cucurbit powdery mildew.

Results

To introduce DNA into P. xanthii conidia, we applied two square-wave pulses of 1.7 kV for 1 ms with an interval of 5 s. We tested these conditions with several plasmids bearing as selective markers hygromycin B resistance (hph), carbendazim resistance (TUB2) or GFP (gfp) under control of endogenous regulatory elements from Aspergillus nidulans, Neurospora crassa or P. xanthii to drive their expression. An in planta selection procedure using the MBC fungicide carbendazim permitted the propagation of transformants onto zucchini cotyledons and avoided the phytotoxicity associated with hygromycin B.

Conclusion

This is the first report on the transformation of P. xanthii and the transformation of powdery mildew fungi using electroporation. Although the transformants are transient, this represents a feasible method for the genetic manipulation of this important group of plant pathogens.     

Effects of Mildew and Nitrogen on Grain Yield of Barley Artifically Infected in the Field

Powdery mildew was artificially introduced into field plotsof barley given two amounts of nitrogen fertilizer. In Tyraand Triumph, grain dry matter and nitrogen yield of healthyplots increased in response to nitrogen fertilizer. In mildewedplots increases in response to nitrogen did not occur in Triumph,despite low levels of mildew infection. Mildew reduced grainyield and nitrogen content at both levels of nitrogen in bothvarieties. Hordewm vulgare L., barley, Erysiphe graminis, powdery mildew, nitrogen fertilizer, grain yield     

The ARP2/3 complex,acting cooperatively with Class I formins,modulates penetration resistance in Arabidopsis against powdery mildew invasion

The actin cytoskeleton regulates an array of diverse cellular activities that support the establishment of plant–microbe interactions and plays a critical role in the execution of plant immunity. However, molecular and cellular mechanisms regulating the assembly and rearrangement of actin filaments (AFs) at plant–pathogen interaction sites remain largely elusive. Here, using live-cell imaging, we show that one of the earliest cellular responses in Arabidopsis thaliana upon powdery mildew attack is the formation of patch-like AF structures beneath fungal invasion sites. The AFs constituting actin patches undergo rapid turnover, which is regulated by the actin-related protein (ARP)2/3 complex and its activator, the WAVE/SCAR regulatory complex (W/SRC). The focal accumulation of phosphatidylinositol-4,5-bisphosphate at fungal penetration sites appears to be a crucial upstream modulator of the W/SRC–ARP2/3 pathway-mediated actin patch formation. Knockout of W/SRC–ARP2/3 pathway subunits partially compromised penetration resistance with impaired endocytic recycling of the defense-associated t-SNARE protein PEN1 and its deposition into apoplastic papillae. Simultaneously knocking out ARP3 and knocking down the Class I formin (AtFH1) abolished actin patch formation, severely impaired the deposition of cell wall appositions, and promoted powdery mildew entry into host cells. Our results demonstrate that the ARP2/3 complex and formins, two actin-nucleating systems, act cooperatively and contribute to Arabidopsis penetration resistance to fungal invasion.

ARP2/3 complex, acting cooperatively with Class I formins, modulates actin patch formation beneath fungal penetration sites, contributing to the penetration resistance of Arabidopsis against powdery mildew invasion.     

Silicon supply in soilless cultivations of zucchini alleviates stress induced by salinity and powdery mildew infections

In the present study, the hypothesis was tested as to whether silicon supplied via the nutrient solution is capable of enhancing the tolerance of hydroponically grown zucchini squash (Cucurbita pepo L. cv. ‘Rival’) to salinity and powdery mildew infections. Two experiments were conducted involving a low (2.2 dS m^?1, 0.8 mM NaCl) and a high salinity level (6.2 dS m^?1, 35 mM NaCl) in combination with a low (0.1 mM) and a high (1.0 mM) Si level in the nutrient solution supplied to the crop. The exposure of the plants to high external salinity restricted significantly the vegetative growth as well as the fruit yield of zucchini due to a reduction of both the number of fruits per plant and the mean fruit weight. However, the inclusion of 1 mM of Si in the salinized nutrient solution mitigated the salinity-associated suppression of both growth and yield. Part of the growth and fruit yield suppression at high salinity was due to restriction of net photosynthesis. The stomatal conductance was also restricted by salinity, whereas the substomatal CO₂ concentration was not affected by the NaCl or Si treatments. The supply of 1 mM of Si via the nutrient solution mitigated the inhibitory effect of salinity on net photosynthesis and this effect was associated with lower Na and Cl translocation to the epigeous plant tissues. Furthermore, the supply of Si via the nutrient solution suppressed appreciably the expansion of a powdery mildew (Podosphaera xanthii) infection in the leaves at both salinity levels. These results indicate that the supply of at least 1 mM of Si via the nutrient solution is capable of enhancing both tolerance to salinity and resistance to powdery mildew in soilless cultivations of zucchini squash.     

A maximum entropy approach to the study of residue-specific backbone angle distributions in α-synuclein,an intrinsically disordered protein

α-Synuclein is an intrinsically disordered protein of 140 residues that switches to an α-helical conformation upon binding phospholipid membranes. We characterize its residue-specific backbone structure in free solution with a novel maximum entropy procedure that integrates an extensive set of NMR data. These data include intraresidue and sequential H^N–H^α and H^N–H^N NOEs, values for ³J_HNHα, ¹J_HαCα, ²J_CαN, and ¹J_CαN, as well as chemical shifts of ¹⁵N, ¹³C^α, and ¹³C′ nuclei, which are sensitive to backbone torsion angles. Distributions of these torsion angles were identified that yield best agreement to the experimental data, while using an entropy term to minimize the deviation from statistical distributions seen in a large protein coil library. Results indicate that although at the individual residue level considerable deviations from the coil library distribution are seen, on average the fitted distributions agree fairly well with this library, yielding a moderate population (20–30%) of the PP_II region and a somewhat higher population of the potentially aggregation-prone β region (20–40%) than seen in the database. A generally lower population of the α_R region (10–20%) is found. Analysis of ¹H–¹H NOE data required consideration of the considerable backbone diffusion anisotropy of a disordered protein.     

Computational and Experimental Studies on β-Sheet Breakers Targeting Aβ1–40 Fibrils

In this work we present and compare the results of extensive molecular dynamics simulations of model systems comprising an Aβ_1–40 peptide in water in interaction with short peptides (β-sheet breakers) mimicking the 17–21 region of the Aβ_1–40 sequence. Various systems differing in the customized β-sheet breaker structure have been studied. Specifically we have considered three kinds of β-sheet breakers, namely Ac-LPFFD-NH₂ and two variants thereof, one obtained by substituting the acetyl group with the sulfonic amino acid taurine (Tau-LPFFD-NH₂) and a second novel one in which the aspartic acid is substituted by an asparagine (Ac-LPFFN-NH₂). Thioflavin T fluorescence, circular dichroism, and mass spectrometry experiments have been performed indicating that β-sheet breakers are able to inhibit in vitro fibril formation and prevent the β sheet folding of portions of the Aβ_1–40 peptide. We show that molecular dynamics simulations and far UV circular dichroism provide consistent evidence that the new Ac-LPFFN-NH₂ β-sheet breaker is more effective than the other two in stabilizing the native α-helix structure of Aβ_1–40. In agreement with these results thioflavin T fluorescence experiments confirm the higher efficiency in inhibiting Aβ_1–40 aggregation. Furthermore, mass spectrometry data and molecular dynamics simulations consistently identified the 17–21 Aβ_1–40 portion as the location of the interaction region between peptide and the Ac-LPFFN-NH₂ β-sheet breaker.     

The measurement of the intrinsic alkaline Bohr effect of various human haemoglobins by isoelectric focusing

We have used isoelectric focusing to measure the differences between the pI values of various normal and mutant human haemoglobins when completely deoxygenated and when fully liganded with CO. It was assumed that the ΔpI(deox.–ox.) values might correspond quantitatively to the intrinsic alkaline Bohr effect, as most of the anionic cofactors of the haemoglobin molecule are `stripped' off during the electrophoretic process. In haemoglobins known to exhibit a normal Bohr coefficient (ΔlogP₅₀/ΔpH) in solutions, the ΔpI(deox.–ox.) values are lower the higher their respective pI(ox.) values. This indicates that for any particular haemoglobin the ΔpI(deox.–ox.) value accounts for the difference in surface charges at the pH of its pI value. This was confirmed by measuring, by the direct-titration technique, the difference in pH of deoxy and fully liganded haemoglobin A₀ (α₂β₂) solutions in conditions approximating those of the isoelectric focusing, i.e. at 5°C and very low concentration of KCl. The variation of the ΔpH(deox.–ox.) curve as a function of pH (ox.) was similar to the isoelectric-focusing curve relating the variation of ΔpI(deox.–ox.) versus pI(ox.) in various haemoglobins with Bohr factor identical with that of haemoglobin A₀. In haemoglobin A₀ the ΔpI(deox.–ox.) value is 0.17 pH unit, which corresponds to a difference of 1.20 positive charges between the oxy and deoxy states of the tetrameric haemoglobin. This value compares favourably with the values of the intrinsic Bohr effect estimated in back-titration experiments. The ΔpI(deox.–ox.) values of mutant or chemically modified haemoglobins carrying an abnormality at the N- or C-terminus of the α-chains are decreased by 30% compared with the ΔpI value measured in haemoglobin A₀. When the C-terminus of the β-chains is altered, as in Hb Nancy (α₂β^{Tyr-145→Asp}₂), we observed a 70% decrease in the ΔpI value compared with that measured in haemoglobin A₀. These values are in close agreement with the estimated respective roles of the two major Bohr groups, Val-1α and His-146β, at the origin of the intrinsic alkaline Bohr effect [Kilmartin, Fogg, Luzzana & Rossi-Bernardi (1973) J. Biol. Chem. 248, 7039–7043; Perutz, Kilmartin, Nishikura, Fogg, Butler & Rollema (1980) J. Mol. Biol. 138, 649–670]. In other mutant haemoglobins it is demonstrated also that the ΔpI(deox.–ox.) value may be decreased or even suppressed when the substitution affects residues involved in the stability of the tetramer. These results support the interpretation proposed by Perutz, Kilmartin, Nishikura, Fogg, Butler & Rollema [(1980), J. Mol. Biol. 138, 649–670] for the mechanism of the alkaline Bohr effect, and also indicate that the transition between the two quaternary configurations is a prerequisite for the full expression of the alkaline Bohr effect.     

Dracula’s ménagerie: A multispecies occupancy analysis of lynx,wildcat, and wolf in the Romanian Carpathians

1. The recovery of terrestrial carnivores in Europe is a conservation success story. Initiatives focused on restoring top predators require information on how resident species may interact with the re‐introduced species as their interactions have the potential to alter food webs, yet such data are scarce for Europe.
2. In this study, we assessed patterns of occupancy and interactions between three carnivore species in the Romanian Carpathians. Romania houses one of the few intact carnivore guilds in Europe, making it an ideal system to assess intraguild interactions and serve as a guide for reintroductions elsewhere.
3. We used camera trap data from two seasons in Transylvanian forests to assess occupancy and co‐occurrence of carnivores using multispecies occupancy models.
4. Mean occupancy in the study area was highest for lynx (Ψ_winter = 0.76 95% CI: 0.42–0.92; Ψ_autumn = 0.71 CI: 0.38–0.84) and wolf (Ψ_winter = 0.60 CI: 0.34–0.78; Ψ_autumn = 0.81 CI: 0.25–0.95) and lowest for wildcat (Ψ_winter = 0.40 CI: 0.19–0.63; Ψ_autumn = 0.52 CI: 0.17–0.78)
5. We found that marginal occupancy predictors for carnivores varied between seasons. We also found differences in predictors of co‐occurrence between seasons for both lynx‐wolf and wildcat‐wolf co‐occurrence. For both seasons, we found that conditional occupancy probabilities of all three species were higher when another species was present.
6. Our results indicate that while there are seasonal differences in predictors of occupancy and co‐occurrence of the three species, co‐occurrence in our study area is high.
7. Terrestrial carnivore recovery efforts are ongoing worldwide. Insights into interspecific relations between carnivore species are critical when considering the depauperate communities they are introduced in. Our work showcases that apex carnivore coexistence is possible, but dependent on protection afforded to forest habitats and their prey base.