Stress-induced flowering

Many plant species can be induced to flower by responding to stress factors. The short-day plants Pharbitis nil and Perilla frutescens var. crispa flower under long days in response to the stress of poor nutrition or low-intensity light. Grafting experiments using two varieties of P. nil revealed that a transmissible flowering stimulus is involved in stress-induced flowering. The P. nil and P. frutescens plants that were induced to flower by stress reached anthesis, fruited and produced seeds. These seeds germinated, and the progeny of the stressed plants developed normally. Phenylalanine ammonialyase inhibitors inhibited this stress-induced flowering, and the inhibition was overcome by salicylic acid (SA), suggesting that there is an involvement of SA in stress-induced flowering. PnFT2, a P. nil ortholog of the flowering gene FLOWERING LOCUS T (FT) of Arabidopsis thaliana, was expressed when the P. nil plants were induced to flower under poor-nutrition stress conditions, but expression of PnFT1, another ortholog of FT, was not induced, suggesting that PnFT2 is involved in stress-induced flowering.Key words: flowering, stress, phenylalanine ammonia-lyase, salicylic acid, FLOWERING LOCUS T, Pharbitis nil, Perilla frutescensFlowering in many plant species is regulated by environmental factors, such as night-length in photoperiodic flowering and temperature in vernalization. On the other hand, a short-day (SD) plant such as Pharbitis nil (synonym Ipomoea nil) can be induced to flower under long days (LD) when grown under poor-nutrition, low-temperature or high-intensity light conditions.^1–9 The flowering induced by these conditions is accompanied by an increase in phenylalanine ammonia-lyase (PAL) activity.¹⁰ Taken together, these facts suggest that the flowering induced by these conditions might be regulated by a common mechanism. Poor nutrition, low temperature and high-intensity light can be regarded as stress factors, and PAL activity increases under these stress conditions.¹¹ Accordingly, we assumed that such LD flowering in P. nil might be induced by stress. Non-photoperiodic flowering has also been sporadically reported in several plant species other than P. nil, and a review of these studies suggested that most of the factors responsible for flowering could be regarded as stress. Some examples of these factors are summarized in 12^–14

Table 1

Some cases of stress-induced flowering

Tomato BRI1 and systemin wound signalling

Brassinosteroids (BRs) are perceived by Brassinosteroid Insensitive 1 (BRI1), that encodes a leucine-rich repeat receptor kinase. Tomato BRI1 has previously been implicated in both systemin and BR signalling. The role of tomato BRI1 in BR signalling was confirmed, however it was found not to be essential for systemin/wound signalling. Tomato roots were shown to respond to systemin but this response varied according to the species and growth conditions. Overall the data indicates that mutants defective in tomato BRI1 are not defective in systemin-induced wound signalling and that systemin perception can occur via a non-BRI1 mechanism.Key words: tomato BRI1, brassinosteroids, systemin, wound signallingBrassinosteroids (BRs) are steroid hormones that are essential for normal plant growth. The most important BR receptor in Arabidopsis is BRASSINOSTERIOD INSENSITIVE 1 (BRI1), a serine/threonine kinase with a predicted extracellular domain of ∼24 leucine-rich repeats (LRRs).^1,2 BRs bind to BRI1 via a steroid-binding domain that includes LRR 21 and a so-called “island” domain.^2,3 In tomato a BRI1 orthologue has been identified that when mutated, as in the curl3 (cu3) mutation, results in BR-insensitive dwarf plants.⁴ Tomato BRI1 has also been purified as a systemin-binding protein.⁵ Systemin is an eighteen amino acid peptide, which is produced by post-translational cleavage of prosystemin. Systemin has been implicated in wound signalling and is able to induce the production of jasmonate, protease inhibitors (PIN) and rapid alkalinization of cell suspensions (reviewed in ref. ⁶).To clarify whether tomato BRI1 was indeed a dual receptor it was important to first confirm its role in BR signalling. Initially this was carried out by genetic complementation of the cu3 mutant phenotype.⁷ Overexpression of tomato BRI1 restored the dwarf phenotype and BR sensitivity and normalized BR levels (

Prion interference with multiple prion isolates

Co-inoculation of prion strains into the same host can result in interference, where replication of one strain hinders the ability of another strain to cause disease. The drowsy (DY) strain of hamster-adapted transmissible mink encephalopathy (TME) extends the incubation period or completely blocks the hyper (HY) strain of TME following intracerebral, intraperitoneal or sciatic nerve routes of inoculation. However, it is not known if the interfering effect of the DY TME agent is exclusive to the HY TME agent by these experimental routes of infection. To address this issue, we show that the DY TME agent can block hamster-adapted chronic wasting disease (HaCWD) and the 263K scrapie agent from causing disease following sciatic nerve inoculation. Additionally, per os inoculation of DY TME agent slightly extends the incubation period of per os superinfected HY TME agent. These studies suggest that prion strain interference can occur by a natural route of infection and may be a more generalized phenomenon of prion strains.Key words: prion diseases, prion interference, prion strainsPrion diseases are fatal neurodegenerative diseases that are caused by an abnormal isoform of the prion protein, PrP^Sc.¹ Prion strains are hypothesized to be encoded by strain-specific conformations of PrP^Sc resulting in strain-specific differences in clinical signs, incubation periods and neuropathology.^2–7 However, a universally agreed upon definition of prion strains does not exist. Interspecies transmission and adaptation of prions to a new host species leads to the emergence of a dominant prion strain, which can be due to selection of strains from a mixture present in the inoculum, or produced upon interspecies transmission.^8,9 Prion strains, when present in the same host, can interfere with each other.Prion interference was first described in mice where a long incubation period strain 22C extended the incubation period of a short incubation period strain 22A following intracerebral inoculation.¹⁰ Interference between other prion strains has been described in mice and hamsters using rodent-adapted strains of scrapie, TME, Creutzfeldt-Jacob disease and Gerstmannn-Sträussler-Scheinker syndrome following intracerebral, intraperitoneal, intravenous and sciatic nerve routes of inoculation.^10–15 We previously demonstrated the detection of PrP^Sc from the long incubation period DY TME agent correlated with its ability to extend the incubation period or completely block the superinfecting short incubation period HY TME agent from causing disease and results in a reduction of HY PrP^Sc levels following sciatic nerve inoculation.¹² However, it is not known if a single long incubation period agent (e.g., DY TME) can interfere with more than one short incubation period agent or if interference can occur by a natural route of infection.To examine the question if one long incubation period agent can extend the incubation period of additional short incubation period agents, hamsters were first inoculated in the sciatic nerve with the DY TME agent 120 days prior to superinfection with the short-incubation period agents HY TME, 263K scrapie and HaCWD.^16–18 The HY TME and 263K scrapie agents have been biologically cloned and have distinct PrP^Sc properties.^19,20 The HaCWD agent used in this study is seventh hamster passage that has not been biologically cloned and therefore will be referred to as a prion isolate. Sciatic nerve inoculations were performed as previously described.^11,12 Briefly, hamsters were inoculated with 103.0 i.c. LD₅₀ of the DY TME agent or equal volume (2 µl of a 1% w/v brain homogenate) of uninfected brain homogenate 120 days prior to superinfection of the same sciatic nerve with either 10^4.6 i.c. LD₅₀ of the HY TME agent, 10^5.2 i.c. LD₅₀ of the HaCWD agent or 10^4.6 i.c. LD₅₀/g 263K scrapie agent (Bartz J, unpublished data).^16,18,21 Animals were observed three times per week for the onset of clinical signs of HY TME, 263K and HaCWD based on the presence of ataxia and hyperexcitability, while the clinical diagnosis of DY TME was based on the appearance of progressive lethargy.^16–18 The incubation period was calculated as the number of days between the onset of clinical signs of the agent strain that caused disease and the inoculation of that strain. The Student''s t-test was used to compare incubation periods.¹² We found that sciatic nerve inoculation of both the HaCWD agent and 263K scrapie agent caused disease with a similar incubation period to animals infected with the HY TME agent (12 In hamsters inoculated with the DY TME agent 120 days prior to superinfection with the HaCWD or 263K agents, the animals developed clinical signs of DY TME with an incubation period that was not different from the DY TME agent control group (12 The PrP^Sc migration properties were consistent with the clinical diagnosis and all co-infected animals had PrP^Sc that migrated similar to PrP^Sc from the DY TME agent infected control animal (Fig. 1, lanes 1–10). This data indicates that the DY TME agent can interfere with more than one isolate and that interference in the CNS may be a more generalized phenomenon of prion strains.Open in a separate windowFigure 1The strain-specific properties of PrP^Sc correspond to the clinical diagnosis of disease. Western blot analysis of 250 µg brain equivalents of proteinase K digested brain homogenate from prion-infected hamsters following intracerebral (i.c.), sciatic nerve (i.sc.) or per os inoculation with either the HY TME (HY), DY TME (DY), 263K scrapie (263K), hamster-adapted CWD (CWD) agents or mock-infected (UN). The unglycoyslated PrP^Sc glycoform of HY TME, 263K scrapie and hamster-adapted CWD migrates at 21 kDa. The unglycosylated PrP^Sc glycoform of DY PrP^Sc migrates at 19 kDa. Migration of 19 and 21 kDa PrP^Sc are indicated by the arrows on the left of the figure. n.a., not applicable.

Table 1

Clinical signs and incubation periods of hamsters inoculated in the sciatic nerve with either the HY TME, HaCWD or 263K scrapie agents, or co-infected with the DY TME agent 120 days prior to superinfection of hamsters with the HY TME, HaCWD or 263K agents

Comparative Analysis of Myxococcus Predation on Soil Bacteria

Predator-prey relationships among prokaryotes have received little attention but are likely to be important determinants of the composition, structure, and dynamics of microbial communities. Many species of the soil-dwelling myxobacteria are predators of other microbes, but their predation range is poorly characterized. To better understand the predatory capabilities of myxobacteria in nature, we analyzed the predation performance of numerous Myxococcus isolates across 12 diverse species of bacteria. All predator isolates could utilize most potential prey species to effectively fuel colony expansion, although one species hindered predator swarming relative to a control treatment with no growth substrate. Predator strains varied significantly in their relative performance across prey types, but most variation in predatory performance was determined by prey type, with Gram-negative prey species supporting more Myxococcus growth than Gram-positive species. There was evidence for specialized predator performance in some predator-prey combinations. Such specialization may reduce resource competition among sympatric strains in natural habitats. The broad prey range of the Myxococcus genus coupled with its ubiquity in the soil suggests that myxobacteria are likely to have very important ecological and evolutionary effects on many species of soil prokaryotes.Predation plays a major role in shaping both the ecology and evolution of biological communities. The population and evolutionary dynamics of predators and their prey are often tightly coupled and can greatly influence the dynamics of other organisms as well (1). Predation has been invoked as a major cause of diversity in ecosystems (11, 12). For example, predators may mediate coexistence between superior and inferior competitors (2, 13), and differential trajectories of predator-prey coevolution can lead to divergence between separate populations (70).Predation has been investigated extensively in higher organisms but relatively little among prokaryotes. Predation between prokaryotes is one of the most ancient forms of predation (27), and it has been proposed that this process may have been the origin of eukaryotic cells (16). Prokaryotes are key players in primary biomass production (44) and global nutrient cycling (22), and predation of some prokaryotes by others is likely to significantly affect these processes. Most studies of predatory prokaryotes have focused on Bdellovibrionaceae species (e.g., see references 51, 55, and 67). These small deltaproteobacteria prey on other Gram-negative cells, using flagella to swim rapidly until they collide with a prey cell. After collision, the predator cells then enter the periplasmic space of the prey cell, consume the host cell from within, elongate, and divide into new cells that are released upon host cell lysis (41). Although often described as predatory, the Bdellovibrionaceae may also be considered to be parasitic, as they typically depend (apart from host-independent strains that have been observed [60]) on the infection and death of their host for their reproduction (47).In this study, we examined predation among the myxobacteria, which are also deltaproteobacteria but constitute a monophyletic clade divergent from the Bdellovibrionaceae (17). Myxobacteria are found in most terrestrial soils and in many aquatic environments as well (17, 53, 74). Many myxobacteria, including the model species Myxococcus xanthus, exhibit several complex social traits, including fruiting body formation and spore formation (14, 18, 34, 62, 71), cooperative swarming with two motility systems (64, 87), and group (or “wolf pack”) predation on both bacteria and fungi (4, 5, 8, 9, 15, 50). Using representatives of the genus Myxococcus, we tested for both intra- and interspecific variation in myxobacterial predatory performance across a broad range of prey types. Moreover, we examined whether prey vary substantially in the degree to which they support predatory growth by the myxobacteria and whether patterns of variation in predator performance are constant or variable across prey environments. The latter outcome may reflect adaptive specialization and help to maintain diversity in natural populations (57, 59).Although closely related to the Bdellovibrionaceae (both are deltaproteobacteria), myxobacteria employ a highly divergent mode of predation. Myxobacteria use gliding motility (64) to search the soil matrix for prey and produce a wide range of antibiotics and lytic compounds that kill and decompose prey cells and break down complex polymers, thereby releasing substrates for growth (66). Myxobacterial predation is cooperative both in its “searching” component (6, 31, 82; for details on cooperative swarming, see reference 64) and in its “handling” component (10, 29, 31, 32), in which secreted enzymes turn prey cells into consumable growth substrates (56, 83). There is evidence that M. xanthus employs chemotaxis-like genes in its attack on prey cells (5) and that predation is stimulated by close contact with prey cells (48).Recent studies have revealed great genetic and phenotypic diversity within natural populations of M. xanthus, on both global (79) and local (down to centimeter) scales (78). Phenotypic diversity includes variation in social compatibility (24, 81), the density and nutrient thresholds triggering development (33, 38), developmental timing (38), motility rates and patterns (80), and secondary metabolite production (40). Although natural populations are spatially structured and both genetic diversity and population differentiation decrease with spatial scale (79), substantial genetic diversity is present even among centimeter-scale isolates (78). No study has yet systematically investigated quantitative natural variation in myxobacterial predation phenotypes across a large number of predator genotypes.Given the previous discovery of large variation in all examined phenotypes, even among genetically extremely similar strains, we anticipated extensive predatory variation as well. Using a phylogenetically broad range of prey, we compared and contrasted the predatory performance of 16 natural M. xanthus isolates, sampled from global to local scales, as well as the commonly studied laboratory reference strain DK1622 and representatives of three additional Myxococcus species: M. flavescens (86), M. macrosporus (42), and M. virescens (63) (Table ​(Table1).1). In particular, we measured myxobacterial swarm expansion rates on prey lawns spread on buffered agar (31, 50) and on control plates with no nutrients or with prehydrolyzed growth substrate.

TABLE 1.

List of myxobacteria used, with geographical origin

Evidence for a New Avian Paramyxovirus Serotype 10 Detected in Rockhopper Penguins from the Falkland Islands

The biological, serological, and genomic characterization of a paramyxovirus recently isolated from rockhopper penguins (Eudyptes chrysocome) suggested that this virus represented a new avian paramyxovirus (APMV) group, APMV10. This penguin virus resembled other APMVs by electron microscopy; however, its viral hemagglutination (HA) activity was not inhibited by antisera against any of the nine defined APMV serotypes. In addition, antiserum generated against this penguin virus did not inhibit the HA of representative viruses of the other APMV serotypes. Sequence data produced using random priming methods revealed a genomic structure typical of APMV. Phylogenetic evaluation of coding regions revealed that amino acid sequences of all six proteins were most closely related to APMV2 and APMV8. The calculation of evolutionary distances among proteins and distances at the nucleotide level confirmed that APMV2, APMV8, and the penguin virus all were sufficiently divergent from each other to be considered different serotypes. We propose that this isolate, named APMV10/penguin/Falkland Islands/324/2007, be the prototype virus for APMV10. Because of the known problems associated with serology, such as antiserum cross-reactivity and one-way immunogenicity, in addition to the reliance on the immune response to a single protein, the hemagglutinin-neuraminidase, as the sole base for viral classification, we suggest the need for new classification guidelines that incorporate genome sequence comparisons.Viruses from the Paramyxoviridae family have caused disease in humans and animals for centuries. Over the last 40 years, many paramyxoviruses isolated from animals and people have been newly described (16, 17, 22, 29, 31, 32, 36, 42, 44, 46, 49, 58, 59, 62-64). Viruses from this family are pleomorphic, enveloped, single-stranded, nonsegmented, negative-sense RNA viruses that demonstrate serological cross-reactivity with other paramyxoviruses related to them (30, 46). The subfamily Paramyxovirinae is divided into five genera: Respirovirus, Morbillivirus, Rubulavirus, Henipavirus, and Avulavirus (30). The Avulavirus genus contains nine distinct avian paramyxovirus (APMV) serotypes (Table ​(Table1),1), and information on the discovery of each has been reported elsewhere (4, 6, 7, 9, 12, 34, 41, 50, 51, 60, 68).

TABLE 1.

Characteristics of prototype viruses APMV1 to APMV9 and the penguin virus

Indirect effects of tending ants on holm oak volatiles and acorn quality

The indirect effect of ants on plants through their mutualism with honeydew-producing insects has been extensively investigated. Honeydew-producing insects that are tended by ants impose a cost on plant fitness and health by reducing seed production and/or plant growth. This cost is associated with sap intake and virus transmissions but may be overcompesated by tending ants if they deter or prey on hebivorous insects. The balance between cost and benefits depends on the tending ant species. In this study we report other indirect effects on plants of the mutualism between aphids and ants. We have found that two Lasius ant species, one native and the other invasive, may change the composition of volatile organic compounds (VOCs) of the holm oak (Quercus ilex) blend when they tend the aphid Lachnus roboris. The aphid regulation of its feeding and honeydew production according to the ant demands was proposed as a plausible mechanism that triggers changes in VOCs. Additionally, we now report here that aphid feeding, which is located most of the time on acorns cap or petiole, significantly increased the relative content of linolenic acid in acorns from holm oak colonized by the invasive ant. This acid is involved in the response of plants to insect herbivory as a precursor or jasmonic acid. No effect was found on acorn production, germination or seedlings quality. These results suggest that tending-ants may trigger the physiological response of holm oaks involved in plant resistance toward aphid herbivory and this response is ant species-dependent.Key words: tended aphid, invasive ants, linolenic acid, jasmonic acid, monoterpene emissionsTo achieve an indirect effect it is necessary to have a minimun of three species, two focal species that interact directly and an associate species whose presence promotes an indirect effect on one or both focal species. In general, indirect effects of a third species are defined by how and to what degree a pairwise species interaction is influenced by the presence and density of this third species.¹ There are several examples of interactions presenting indirect effects: apparent competition,¹ facilitation,² tri-trophic level interactions,³ cascading effects⁴ and exploitative competition. 5 But, indirect effects have been studied most extensively in the context of trophic cascades when top predators are removed⁶ or added⁷ and in the context of mutualisms.^8–10 Usually, indirect effects are investigated as changes in abundance of the focal species occur. However, indirect effects may result in biologically significant changes in a species that are not reflected only to its abundance.¹¹ There are many examples of changes in physiology, behavior, morphology and/or genotypic composition of the focal species.^11,12 These changes on density and/or morphological, physiological and behavioral traits of the focal species are not mutually exclusive, and all can act at the same time.¹³ The magnitude and direction of both direct and indirect effects should influence the relative resilience of communities to perturbation, which in turn will affect species coexistence and community evolution.¹⁴ In this regard, indirect effects had been postulated as one of the main forces structuring communities² and shaping the evolution of communities.¹⁴In terrestrial communities ants interact with plants both directly and indirectly. They can disperse or consume seeds, feed from specialized plant structures such as food bodies and extrafloral nectaries, act as or deter pollinitators, prey on herbivorous insects and/or develop mutualisms with honeydew-producing insects indirectly modifying plant fitness.^15–17 Additionally, through their nesting activities in soil, ants increase soil nutrient content available to plants, may change water infiltration and soil holding-capacity and modify biodiversity and abundance of soil organisms related to the decomposition process.^18,19 As a consequence of their activities, ants may thus change behavior, density, physiology or fitness of other species.^12,22,23 In the case of ants that tend honeydew-producing insects, evidence shows that their attention may change some traits of insect life history, 22 their abundance or physiology.¹⁸ For the plant, the net outcome of the mutualism between ants and honeydew-producing insects will depend on the balance between the costs for plant fitness via consumption of plant sap and transmission of plant pathogens and the benefit of ants deterring herbivorous insects.^18,23 As a consequence, plant seed production, pod production or even plant growth may decrease when the cost of honeydew-producing insects exceed the benefit provided by tending ants.^18,23Recently, we have described the changes that two tending ant species may exert indirectly on monoterpene emissions of holm oak (Quercus ilex) saplings through its mutualism with Lachnus roboris aphids.²⁴ One of these tending ant species was Lasius neglectus, an invasive ant species that displaces the local ant Lasius grandis. We found that aphids feeding on holm oak increased the emission of total volatile organic carbon (VOCs) by 31%. In particular, aphids feeding elicited the emission of a new monoterpene, Δ³-carene, and increased the emission of myrcene (mean ± SE; sapling alone: 0.105 ± 0.011 µg g⁻¹ h⁻¹; sapling plus not tended aphid: 0.443 ± 0.057 µg g⁻1 h⁻1) and γ-terpinene (sapling alone: 0.0013 ± 0.0001; sapling plus not tended aphid: 0.0122 ± 0.0022 µg g⁻1 h⁻1) (Mann-Whitney, sapling alone vs. sapling plus not tended aphids, U_4,4 = 0, p < 0.05 for both compounds). Changes of VOC emission in response to aphid infestation were noticed also in boreal trees.²⁴ When the aphids became tended by the invasive ant, L. neglectus, VOCs emissions increased only 19% because myrcene, the main compound of the blend, decreased significantly (25 When our data was recalculated on leaf area basis (nmol m⁻² s⁻¹), the general pattern was the same independently of the units, but the differences among treatments were not statistically significant (26 These slight differences in the statitiscal significance of the differences of VOC emissions depending on the reference unit may be due to differences in leaf morphology, i.e., changes of leaf area and mass. However, in our study, all holm oaks showed a similar leaf morphology among treatments (Kruskal-Wallis, leaf mass: H_3,20 = 2.16, p = 0.53; leaf area: H_3,20 = 2.64, p = 0.45) (24^,27 This lack of consistence of aphid effect on leaf area and mass limits the development of a clear pattern linking aphids feeding, leaf area or mass and VOC emissions. On the other hand, to achieve statistical significance of emitted VOCs among treatments, values should differ strongly given the high variability of VOC emission within treatments.²⁶ Under this scenario, we recommend giving the values of leaf morphology and to give VOC emissions on both unit bases to facilite comparisons among different studies.

Table 1

Means and standard error of the emission rates of the main compounds emitted by Quercus ilex saplings (n = 4 for T1 and T2 and n = 8 for T3) infested with untended aphids (T1) or infested with aphids tended by the native ant Lasius grandis (T2) or by the invasive ant Lasius neglectus (T3)

Genome-wide analysis of lipoxygenase gene family in Arabidopsis and rice

The enzymes called lipoxygenases (LOXs) can dioxygenate unsaturated fatty acids, which leads to lipoperoxidation of biological membranes. This process causes synthesis of signaling molecules and also leads to changes in cellular metabolism. LOXs are known to be involved in apoptotic (programmed cell death) pathway, and biotic and abiotic stress responses in plants. Here, the members of LOX gene family in Arabidopsis and rice are identified. The Arabidopsis and rice genomes encode 6 and 14 LOX proteins, respectively, and interestingly, with more LOX genes in rice. The rice LOXs are validated based on protein alignment studies. This is the first report wherein LOXs are identified in rice which may allow better understanding the initiation, progression and effects of apoptosis, and responses to bitoic and abiotic stresses and signaling cascades in plants.Key words: apoptosis, biotic and abiotic stresses, genomics, jasmonic acid, lipidsLipoxygenases (linoleate:oxygen oxidoreductase, EC 1.13.11.-; LOXs) catalyze the conversion of polyunsaturated fatty acids (lipids) into conjugated hydroperoxides. This process is called hydroperoxidation of lipids. LOXs are monomeric, non-heme and non-sulfur, but iron-containing dioxygenases widely expressed in fungi, animal and plant cells, and are known to be absent in prokaryotes. However, a recent finding suggests the existence of LOX-related genomic sequences in bacteria but not in archaea.¹ The inflammatory conditions in mammals like bronchial asthama, psoriasis and arthritis are a result of LOXs reactions.² Further, several clinical conditions like HIV-1 infection,³ disease of kidneys due to the activation of 5-lipoxygenase,^4,5 aging of the brain due to neuronal 5-lipoxygenase⁶ and atherosclerosis⁷ are mediated by LOXs. In plants, LOXs are involved in response to biotic and abiotic stresses.⁸ They are involved in germination⁹ and also in traumatin and jasmonic acid biochemical pathways.^10,11 Studies on LOX in rice are conducted to develop novel strategies against insect pests¹² in response to wounding and insect attack,¹³ and on rice bran extracts as functional foods and dietary supplements for control of inflammation and joint health.¹⁴ In Arabidopsis, LOXs are studied in response to natural and stress-induced senescence,¹⁵ transition to flowering,¹⁶ regulation of lateral root development and defense response.¹⁷The arachidonic, linoleic and linolenic acids can act as substrates for different LOX isozymes. A hydroperoxy group is added at carbons 5, 12 or 15, when arachidonic acid is the substrate, and so the LOXs are designated as 5-, 12- or 15-lipoxygenases. Sequences are available in the database for plant lipoxygenases (EC:1.13.11.12), mammalian arachidonate 5-lipoxygenase (EC:1.13.11.34), mammalian arachidonate 12-lipoxygenase (EC:1.13.11.31) and mammalian erythroid cell-specific 15-lipoxygenase (EC:1.13.11.33). The prototype member for LOX family, LOX-1 of Glycine max L. (soybean) is a 15-lipoxygenase. The LOX isoforms of soybean (LOX-1, LOX-2, LOX-3a and LOX-3b) are the most characterized of plant LOXs.¹⁸ In addition, five vegetative LOXs (VLX-A, -B, -C, -D, -E) are detected in soybean leaves.¹⁹ The 3-dimensional structure of soybean LOX-1 has been determined.^20,21 LOX-1 was shown to be made of two domains, the N-terminal domain-I which forms a β-barrel of 146 residues, and a C-terminal domain-II of bundle of helices of 693 residues²¹ (Fig. 1). The iron atom was shown to be at the centre of domain-II bound by four coordinating ligands, of which three are histidine residues.²²Open in a separate windowFigure 1Three-dimensional structure of soybean lipoxygenase L-1. The domain I (N-terminal) and domain II (C-terminal) are indicated. The catalytic iron atom is embedded in domain II (PDB ID-1YGE).²¹This article describes identification of LOX genes in Arabidopsis and rice. The Arabidopsis genome encodes for six LOX proteins²³ (www.arabidopsis.org) (LocusAnnotationNomenclatureA^*B^*C^*AT1G55020lipoxygenase 1 (LOX1)LOX185998044.45.2049AT1G17420lipoxygenase 3 (LOX3)LOX3919103725.18.0117AT1G67560lipoxygenase family proteinLOX4917104514.68.0035AT1G72520lipoxygenase, putativeLOX6926104813.17.5213AT3G22400lipoxygenase 5 (LOX5)LOX5886101058.86.6033AT3G45140lipoxygenase 2 (LOX2)LOX2896102044.75.3177Open in a separate window^*A, amino acids; B, molecular weight; C, isoelectric point.Interestingly, the rice genome (rice.plantbiology.msu.edu) encodes for 14 LOX proteins as compared to six in Arabidopsis (​and22). Of these, majority of them are composed of ∼790–950 aa with the exception for loci, LOC_Os06g04420 (126 aa), LOC_Os02g19790 (297 aa) and LOC_Os12g37320 (359 aa) (Fig. 2).Open in a separate windowFigure 2Protein alignment of rice LOXs and vegetative lipoxygenase, VLX-B,²⁸ a soybean LOX (AA B67732). The 14 rice LOCs are indicated on left and sequence position on right. Gaps are included to improve alignment accuracy. Figure was generated using ClustalX program.

Table 2

Genes encoding lipoxygenases in rice

Natural Infection of Burkholderia pseudomallei in an Imported Pigtail Macaque (Macaca nemestrina) and Management of the Exposed Colony

Identification of the select agent Burkholderia pseudomallei in macaques imported into the United States is rare. A purpose-bred, 4.5-y-old pigtail macaque (Macaca nemestrina) imported from Southeast Asia was received from a commercial vendor at our facility in March 2012. After the initial acclimation period of 5 to 7 d, physical examination of the macaque revealed a subcutaneous abscess that surrounded the right stifle joint. The wound was treated and resolved over 3 mo. In August 2012, 2 mo after the stifle joint wound resolved, the macaque exhibited neurologic clinical signs. Postmortem microbiologic analysis revealed that the macaque was infected with B. pseudomallei. This case report describes the clinical evaluation of a B. pseudomallei-infected macaque, management and care of the potentially exposed colony of animals, and protocols established for the animal care staff that worked with the infected macaque and potentially exposed colony. This article also provides relevant information on addressing matters related to regulatory issues and risk management of potentially exposed animals and animal care staff.Abbreviations: CDC, Centers for Disease Control and Prevention; IHA, indirect hemagglutination assay; PEP, postexposure prophylacticBurkholderia pseudomallei, formerly known as Pseudomonas pseudomallei, is a gram-negative, aerobic, bipolar, motile, rod-shaped bacterium. B. pseudomallei infections (melioidosis) can be severe and even fatal in both humans and animals. This environmental saprophyte is endemic to Southeast Asia and northern Australia, but it has also been found in other tropical and subtropical areas of the world.^7,22,32,42 The bacterium is usually found in soil and water in endemic areas and is transmitted to humans and animals primarily through percutaneous inoculation, ingestion, or inhalation of a contaminated source.^{8, 22,28,32,42} Human-to-human, animal-to-animal, and animal-to-human spread are rare.^8,32 In December 2012, the National Select Agent Registry designated B. pseudomallei as a Tier 1 overlap select agent.³⁹ Organisms classified as Tier 1 agents present the highest risk of deliberate misuse, with the most significant potential for mass casualties or devastating effects to the economy, critical infrastructure, or public confidence. Select agents with this status have the potential to pose a severe threat to human and animal health or safety or the ability to be used as a biologic weapon.³⁹Melioidosis in humans can be challenging to diagnose and treat because the organism can remain latent for years and is resistant to many antibiotics.^12,37,41 B. pseudomallei can survive in phagocytic cells, a phenomenon that may be associated with latent infections.^19,38 The incubation period in naturally infected animals ranges from 1 d to many years, but symptoms typically appear 2 to 4 wk after exposure.^13,17,35,38 Disease generally presents in 1 of 2 forms: localized infection or septicemia.²² Multiple methods are used to diagnose melioidosis, including immunofluorescence, serology, and PCR analysis, but isolation of the bacteria from blood, urine, sputum, throat swabs, abscesses, skin, or tissue lesions remains the ‘gold standard.’^9,22,40,42 The prognosis varies based on presentation, time to diagnosis, initiation of appropriate antimicrobial treatment, and underlying comorbidities.^7,28,42 Currently, there is no licensed vaccine to prevent melioidosis.There are several published reports of naturally occurring melioidosis in a variety of nonhuman primates (NHP; 2,10,13,17,25,30,31,35 The first reported case of melioidosis in monkeys was recorded in 1932, and the first published case in a macaque species was in 1966.³⁰ In the United States, there have only been 7 documented cases of NHP with B. pseudomallei infection.^2,13,17 All of these cases occurred prior to the classification of B. pseudomallei as a select agent. Clinical signs in NHP range from subclinical or subacute illness to acute septicemia, localized infection, and chronic infection. NHP with melioidosis can be asymptomatic or exhibit clinical signs such as anorexia, wasting, purulent drainage, subcutaneous abscesses, and other soft tissue lesions. Lymphadenitis, lameness, osteomyelitis, paralysis and other CNS signs have also been reported.^{2,7,10,22,28,32} In comparison, human''s clinical signs range from abscesses, skin ulceration, fever, headache, joint pain, and muscle tenderness to abdominal pain, anorexia, respiratory distress, seizures, and septicemia.^7,9,21,22

Table 1.

Summary of reported cases of naturally occurring Burkholderia pseudomalleiinfections in nonhuman primates

Arabidopsis thaliana overexpressing glycolate oxidase in chloroplasts: H2O2-induced changes in primary metabolic pathways

Reactive oxygen species (ROS) represent both toxic by-products of aerobic metabolism as well as signaling molecules in processes like growth regulation and defense pathways. The study of signaling and oxidative-damage effects can be separated in plants expressing glycolate oxidase in the plastids (GO plants), where the production of H₂O₂ in the chloroplasts is inducible and sustained perturbations can reproducibly be provoked by exposing the plants to different ambient conditions. Thus, GO plants represent an ideal non-invasive model to study events related to the perception and responses to H₂O₂ accumulation. Metabolic profiling of GO plants indicated that under high light a sustained production of H₂O₂ imposes coordinate changes on central metabolic pathways. The overall metabolic scenario is consistent with decreased carbon assimilation, which results in lower abundance of glycolytic and tricarboxylic acid cycle intermediates, while simultaneously amino acid metabolism routes are specifically modulated. The GO plants, although retarded in growth and flowering, can complete their life cycle indicating that the reconfiguration of the central metabolic pathways is part of a response to survive and thus, to adapt to stress conditions imposed by the accumulation of H₂O₂ during the light period.Key words: Arabidopsis thaliana, H₂O₂, oxidative stress, reactive oxygen species, signalingReactive oxygen species (ROS) are key molecules in the regulation of plant development, stress responses and programmed cell death. Depending on the identity of ROS species or its subcellular production site, different cellular responses are provoked.¹ To assess the effects of metabolically generated H₂O₂ in chloroplasts, we have recently generated Arabidopsis plants in which the peroxisomal GO was targeted to chloroplasts.² The GO overexpressing plants (GO plants) show retardation in growth and flowering time, features also observed in catalase, ascorbate peroxidase and MnSOD deficient mutants.^3–5 The analysis of GO plants indicated that H₂O₂ is responsible for the observed phenotype. GO plants represent an ideal non-invasive model system to study the effects of H₂O₂ directly in the chloroplasts because H₂O₂ accumulation can be modulated by growing the plants under different ambient conditions. By this, growth under low light or high CO₂ concentrations minimizes the oxygenase activity of RubisCO and thus the flux through GO whereas the exposition to high light intensities enhances photorespiration and thus the flux through GO.Here, we explored the impact of H₂O₂ production on the primary metabolism of GO plants by assessing the relative levels of various metabolites by gas chromatography coupled to mass spectrometry (GC-MS)⁶ in rosettes of plants grown at low light (30 µmol quanta m⁻² s⁻¹) and after exposing the plants for 7 h to high light (600 µmol quanta m⁻² s⁻¹). The results obtained for the GO5 line are shown in After 1 h at 30 µEAfter 7 h at 600 µEAlanine0.88 ± 0.052.83 ± 0.68Asparagine1.39 ± 0.123.64 ± 0.21Aspartate0.88 ± 0.031.65 ± 0.10GABA1.14 ± 0.051.13 ± 0.05Glutamate0.97 ± 0.041.51 ± 0.07Glutamine1.06 ± 0.111.87 ± 0.06Glycine1.23 ± 0.070.30 ± 0.02Isoleucine3.52 ± 0.403.00 ± 0.15Leucine1.36 ± 0.220.57 ± 0.06Lysine1.49 ± 0.130.38 ± 0.02Methionine0.96 ± 0.054.54 ± 0.51Phenylalanine0.95 ± 0.030.94 ± 0.04Proline1.32 ± 0.221.60 ± 0.13Serine1.05 ± 0.041.49 ± 0.15Threonine4.74 ± 0.175.51 ± 0.34Valine0.91 ± 0.130.29 ± 0.02Citrate/Isocitrate0.65 ± 0.020.64 ± 0.022-oxoglutarate0.95 ± 0.110.76 ± 0.05Succinate0.78 ± 0.040.72 ± 0.02Fumarate0.64 ± 0.030.31 ± 0.01Malate0.74 ± 0.030.60 ± 0.02Pyruvate1.19 ± 0.280.79 ± 0.04Ascorbate1.13 ± 0.142.44 ± 0.45Galactonate-γ-lactone1.81 ± 0.401.62 ± 0.28Fructose1.20 ± 0.130.37 ± 0.01Glucose1.38 ± 0.170.30 ± 0.01Mannose0.90 ± 0.271.34 ± 0.28Sucrose1.04 ± 0.070.49 ± 0.02Fructose-6P0.82 ± 0.151.20 ± 0.15Glucose-6P0.87 ± 0.061.25 ± 0.183-PGA1.13 ± 0.110.35 ± 0.02DHAP1.38 ± 0.091.26 ± 0.08Glycerate0.99 ± 0.040.67 ± 0.01Glycerol1.07 ± 0.041.12 ± 0.05Shikimate1.18 ± 0.040.35 ± 0.01Salicylic acid1.04 ± 0.180.66 ± 0.18Open in a separate windowPlants were grown at 30 µmol m⁻² sec⁻¹ (30 µE). The samples were collected 1 h after the onset of the light period and after 7 h of exposure to 600 µmol m⁻² sec⁻¹ (600 µE), respectively. The values are relative to the respective wild-type (each metabolite = 1) and represent means ± SE of four determinations of eight plants. (*) indicates the value is significantly different from the respective wild-type as determined by the Student''s t test (p < 0.05).At the beginning of the light period in low light conditions, some significant deviations in the levels of metabolites tested were observed in GO plants when compared to the wild-type (2 the transgenic GO activity is sufficient to induce a characteristic metabolic phenotype (Fig. 1). The levels of the tricarboxylic acid (TCA) cycle intermediates, citrate/isocitrate, succinate, fumarate and malate were lower in the GO plants (7 In consequence, OAA might not freely enter the TCA cycle and is redirected to the synthesis of Lys, Thr and Ile, which accumulate in the GO plants (Open in a separate windowFigure 1Simplified scheme of the primary metabolism showing the qualitative variations in metabolite abundance in GO plants obtained by GC-MS analysis (2 Blue boxes indicate a significant increase in the content of the particular metabolite compared to the wild-type, while red boxes indicate a significant decrease. Metabolites without boxes have not been determined. The arrows do not always indicate single steps. Adapted from Baxter et al., 2007.High light treatment induced massive changes in the metabolic profile of GO plants (Fig. 1). The OAA-derived amino acids Asp, Asn, Thr, Ile and Met as well as the 2-oxoglutarate-derived amino acids Glu and Gln accumulated. On the contrary, the levels of the Pyr-derived amino acids Val and Leu and the OAA-derived amino acid Lys decreased. A rational explanation for these metabolic changes is difficult to assess, but these changes could be a consequence of a metabolic reconfiguration in response to high light leading to required physiological functions and thus ensuring continued cellular function and survival, e.g., production of secondary metabolites to mitigate photooxidative damage. The higher levels of Glu observed in the GO plants could be attributed to alternative pathways of glyoxylate metabolism that may occur during photorespiration.⁸ It has been shown earlier that isocitrate derived from glyoxylate and succinate is decarboxylated by cytosolic isocitrate dehydrogenase producing 2-oxoglutarate and further glutamate.⁸In GO plants grown under low light conditions (minimized photorespiratory conditions), the levels of Gly were similar to those of the wild-type whereas, after exposure to high light (photorespiratory conditions), the Gly levels were extremely low, indicating that the GO activity diverts a significant portion of flux from the photorespiratory pathway (7 and also the levels of the lipoic acid-containing subunits of the pyruvate- and 2-oxoglutarate dehydrogenases were shown to be significantly reduced under oxidative stress conditions.^9,10 Similarly, the contents of the soluble sugars sucrose, fructose and glucose and those of 3-PGA and glycerate were lower. In addition, the GO plants showed an impairment in the accumulation of starch under high light conditions, a feature that was not observed if the plants were grown under non-photorespiratory conditions.²Together, these results indicate that the low photosynthetic carbon assimilation in the GO plants exposed to high light is most probably due to enhanced photoinhibition,² the repression of genes encoding photosynthetic components by H₂O₂,^11–13 and the direct damage or inhibition of enzyme activities involved in CO₂ assimilation and energy metabolism by H₂O₂.^7,10,14,15 Moreover, Scarpeci and Valle¹³ showed that in plants treated with the superoxid anion radical producing methylviologen (MV) most of the genes involved in phosphorylytic starch degradation, e.g., the trioseP/Pi translocator and genes involved in starch and sucrose synthesis were repressed, while genes involved in hydrolytic starch breakdown and those involved in sucrose degradation were induced. In line with this, the contents of carbohydrates were also lower in MV-treated plants. Together, these observations can also explain the lower growth rates of the GO plants in conditions where the oxygenase activity of RubisCO becomes important and thus, the flux through GO increases.²The levels of shikimate were lower in GO plants (2^,16 and the low levels of substrates available, as anthocyanins are ultimately synthesized from photosynthates and the GO plants showed a diminished photosynthetic performance.²As expected, the levels of ascorbate and its precursor, galactonate-γ-lactone, were enhanced in the GO plants clearly showing the activation of the cellular antioxidant machinery (10 described the metabolic response to oxidative stress of heterotrophic Arabidopsis cells treated with menadione, which also generates superoxide anion radicals. This oxidative stress was shown to induce metabolic inhibition of flux through the TCA cycle and sectors of amino acid metabolism together with a diversion of carbon into the oxidative pentose phosphate pathway.Signaling and oxidative-damage effects are difficult to separate by manipulating the enzymes of antioxidant systems. In this regard, the GO plants represent a challenging inducible model that avoid acclimatory and adaptative effects. Moreover, it is possible to control the H₂O₂ production in the chloroplasts of GO plants without inducing oxidative damage by changing the conditions of growth.² Further exploration of metabolic changes imposed by different ROS at the cellular and whole organ levels will allow to address many intriguing questions on how plants can rearrange metabolism to cope with oxidative stresses.     

A Targeted Multilocus Genotyping Assay for Lineage,Serogroup, and Epidemic Clone Typing of Listeria monocytogenes

A 30-probe assay was developed for simultaneous classification of Listeria monocytogenes isolates by lineage (I to IV), major serogroup (4b, 1/2b, 1/2a, and 1/2c), and epidemic clone (EC) type (ECI, ECIa, ECII, and ECIII). The assay was designed to facilitate rapid strain characterization and the integration of subtype data into risk-based inspection programs.Listeria monocytogenes is a facultative intracellular pathogen that can cause serious invasive illness (listeriosis) in humans and other animals. L. monocytogenes is responsible for over 25% of food-borne-disease-related deaths attributable to known pathogens and is a leading cause of food recalls due to microbial adulteration (12, 21). However, not all L. monocytogenes subtypes contribute equally to human illness, and substantial differences in the ecologies and virulence attributes of different L. monocytogenes subtypes have been identified (9, 13, 14, 23, 24, 33, 35, 36). Among the four major evolutionary lineages of L. monocytogenes, only lineages I and II are commonly isolated from contaminated food and human listeriosis patients (19, 27, 29, 33). Lineage I strains are overrepresented among human listeriosis isolates, particularly those associated with epidemic outbreaks, whereas lineage II strains are overrepresented in foods and the environment (13, 14, 24). Lineage III strains account for approximately 1% of human listeriosis cases but are common among animal listeriosis isolates and appear to be a host-adapted group that is poorly adapted to food-processing environments (6, 34-36). The ecological and virulence attributes of lineage IV are poorly understood, as this lineage is rare and was only recently described based on a small number of strains (19, 26, 29, 33).L. monocytogenes is differentiated into 13 serotypes; however, four major serogroups (4b, 1/2b, 1/2a, and 1/2c) from within lineages I and II account for more than 98% of human and food isolates (16, 31). Serogroups refer to evolutionary complexes typified by a predominant serotype but which include very rare serotypes that represent minor evolutionary variants (7, 9, 33). Phylogenetic analyses have indicated that rare serotypes may have evolved recently, or even multiple times, from one of the major serotypes (9), and numerous molecular methods fail to discriminate minor serotypes as independent groups (1, 4, 7, 9, 18, 22, 33, 38, 39). Serotyping is one of the most common methods for L. monocytogenes subtyping, and serogroup classifications are a useful component of strain characterization because ecotype divisions appear largely congruent with serogroup distinctions (16, 34). Serogroup 4b strains are of particular public health concern because contamination with these strains appears to increase the probability that a ready-to-eat (RTE) food will be implicated in listeriosis (16, 28). Serogroup 4b strains account for approximately 40% of sporadic listeriosis and also are responsible for the majority of listeriosis outbreaks despite being relatively rare contaminants of food products (9, 13, 17, 30, 34). In addition, serogroup 4b strains are associated with more severe clinical presentations and higher mortality rates than other serogroups (11, 16, 20, 31, 34). Serogroups 1/2a and 1/2b are overrepresented among food isolates but also contribute significantly to human listeriosis, whereas serogroup 1/2c rarely causes human illness and may pose a lower risk of listeriosis for humans (16). Serogroup-specific differences in association with human listeriosis are consistent with the prevalence of virulence-attenuating mutations in inlA within these serogroups (32, 34); however, a number of additional factors likely contribute to these differences.Four previously described epidemic clones (ECs; ECI, ECIa, ECII, and ECIII) of L. monocytogenes have been implicated in numerous listeriosis outbreaks and have contributed significantly to sporadic illness (15, 34). ECI, ECIa, and ECII are distinct groups within serogroup 4b that were each responsible for repeated outbreaks of listeriosis in the United States and Europe. ECIII is a lineage II clone of serotype 1/2a that persisted in the same processing facility for more than a decade prior to causing a multistate outbreak linked to contaminated turkey (15, 25). While there has been speculation that epidemic clones possess unique adaptations that explain their frequent involvement in listeriosis outbreaks (9, 34, 37), it is not clear that epidemic clones are more virulent than other strains with the same serotype. However, contamination of RTE food with EC strains would be cause for increased concern due to the previous involvement of these clones in major outbreaks of listeriosis (16).As a result of the L. monocytogenes subtype-specific differences in ecology, virulence, and association with human illness, molecular subtyping technologies have the potential to inform assessments of relative risk and to improve risk-based inspection programs. The objective of the present study was to develop a single assay for rapid and accurate classification of L. monocytogenes isolates by lineage, major serogroup, and epidemic clone in order to facilitate strain characterization and the integration of subtype data into inspection programs that are based on assessment of relative risk.A database of more than 5.3 Mb of comparative DNA sequences from 238 L. monocytogenes isolates (9, 33-35) was scanned for single nucleotide polymorphisms that could be used to differentiate lineages, major serogroups, and epidemic clones via a targeted multilocus genotyping (TMLGT) approach. The acronym TMLGT is used to distinguish this approach from previously published multilocus genotyping (MLGT) assays that were lineage specific and designed for haplotype discrimination (9, 33). To provide for simultaneous interrogation of the selected polymorphisms via TMLGT, six genomic regions (Table ​(Table1)1) were coamplified in a multiplex PCR. While the previous MLGT assays were based on three lineage-specific multiplexes and required prior identification of lineage identity, TMLGT was designed to target variation across all of the lineages simultaneously and is based on a unique set of amplicons. PCR was performed in 50-μl volumes with 1× High Fidelity PCR buffer (Invitrogen Life Technologies), 2 mM MgSO₄, 100 μM deoxynucleoside triphosphate (dNTP), 300 nM primer, 1.5 U Platinum Taq high-fidelity DNA polymerase (Invitrogen Life Technologies), and 100 ng of genomic DNA. PCR consisted of an initial denaturation of 90 s at 96°C, followed by 40 cycles of 30 s at 94°C, 30 s at 50°C, and 90 s at 68°C. Amplification products were purified using Montage PCR cleanup filter plates (Millipore) and served as a template for allele-specific primer extension (ASPE) reactions utilizing subtype-specific probes.

TABLE 1.

Primers used in multiplex amplification for the TMLGT assay