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 (

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

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

Decorin regulates endothelial cell-matrix interactions during angiogenesis

Interactions between endothelial cells and the surrounding extracellular matrix are continuously adapted during angiogenesis, from early sprouting through to lumen formation and vessel maturation. Regulated control of these interactions is crucial to sustain normal responses in this rapidly changing environment, and dysfunctional endothelial cell behaviour results in angiogenic disorders. The proteoglycan decorin, an extracellular matrix component, is upregulated during angiogenesis. While it was shown previously that the absence of decorin leads to dysregulated angiogenesis in vivo, the molecular mechanisms were not clear. These abnormal endothelial cell responses have been attributed to indirect effects of decorin; however, our recent data provides evidence that decorin directly regulates endothelial cell-matrix interactions. This data will be discussed in conjunction with findings from previous studies, to better understand the role of this proteoglycan in angiogenesis.Key words: decorin, angiogenesis, motility, α2β1 integrin, insulin-like growth factor I receptor, Rac GTPaseLed by appropriate cues, the vascular system undergoes postnatal remodelling (angiogenesis), to maintain tissue homeostasis. Thus while much of the mature endothelium is quiescent, locally activated endothelial cells re-enter the cell cycle, and assume a motile phenotype essential for sprouting and neo-vessel formation. Concomitantly, the surrounding extracellular matrix (ECM) is significantly altered through de novo protein expression, deposition of plasma components and protease-mediated degradation. The latter liberates cryptic binding sites and sequestered growth factors in addition to intact and degraded ECM components, which themselves possess pro- and anti-angiogenic signalling properties. For supported blood flow, endothelium quiescence and integrity is re-established, and the ECM is organized into mature, cross-linked networks. In short, endothelial cells regulate ECM synthesis, assembly and turnover while the structure and composition of ECM in turn influences cellular phenotype. The ECM therefore, plays a critical role in control of endothelial cell behaviour during angiogenesis.Decorin is a member of the small leucine-rich repeat proteoglycan (SLRP) family, which was first discovered ‘decorating’ collagen I fibrils and was subsequently shown to regulate fibrillogenesis.^1,2 Both the protein core and the single, covalently attached glycosaminoglycan (GAG) moieties of decorin are involved in this function, the relevance of which is demonstrated by the phenotype of the decorin null mouse, which exhibits loose, fragile skin due to dysregulated fibrillogenesis.² Interestingly, a role for decorin in postnatal angiogenesis was also revealed by studies in the decorin null background. Corneal neoangiogenesis was reduced.³ Conversely, neo-angiogenesis was enhanced during dermal wound healing, although surprisingly this led to delayed wound closure.⁴ In this case, skin fragility due to the absence of decorin may have hindered wound closure, despite an increased blood supply. It is apparent however, that decorin plays a role in inflammation-associated angiogenesis. Indeed, endothelial cells undergoing angiogenic morphogenesis in this environment express decorin, while quiescent endothelial cells do not,^3–6 indicating that decorin modulates endothelial cell behaviour specifically during inflammatory-associated remodelling of the vascular system.To understand decorin effects on angiogenic morphogenesis within a minimalist environment, various in vitro models of angiogenesis have been employed (6 Similarly, decorin expression enhanced tube formation on matrigel,⁸ but in other studies utilising this substrate was found to either have no influence⁹ or to inhibit tubulogenesis induced by growth factors.¹⁰ In yet another study, decorin inhibited tube formation when presented as a substrate prior to addition of collagen I.⁷ These contrasting observations may reflect the importance of the micro-environment within which decorin is presented. Alternatively, controversial results could result from different sources of decorin since cell types differ in their post-translational modifications of the GAG moiety. Hence, varying length or sulfation patterns of GAG chains may account for different biological activities of decorin. Discrepancies can also be explained as artefacts due to different purification protocols, such as when denaturing conditions are used to extract decorin from tissue. Taken together however, these observations suggest that decorin is neither a pro- nor an anti-angiogenic factor per se, but rather a regulator of angiogenesis, dependent on local cues for different activities. Further, that decorin is capable of both enhancing and inhibiting tubulogenesis may suggest a role in balancing vessel regression versus persistence. Immature vessels have a period of plasticity prior to maturation, during which they can be remodelled, and either regress, or given the appropriate signals, proceed to maturity.¹¹ As a modulator of tube formation, it is tempting to speculate that decorin could influence the switch from immature to mature vessels, favouring one or the other in conjunction with signals from the local environment.

Table 1

Summary of the key functions of decorin in controlling cell behaviour

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

Influence of polyploidy on insect herbivores of native and invasive genotypes of Solidago gigantea (Asteraceae)

Herbivores are sensitive to the genetic structure of plant populations, as genetics underlies plant phenotype and host quality. Polyploidy is a widespread feature of angiosperm genomes, yet few studies have examined how polyploidy influences herbivores. Introduction to new ranges, with consequent changes in selective regimes, can lead to evolution of changes in plant defensive characteristics and also affect herbivores. Here, we examine how insect herbivores respond to polyploidy in Solidago gigantea, using plants derived from both the native range (USA) and introduced range (Europe). S. gigantea has three cytotypes in the US, with two of these present in Europe. We performed bioassays with generalist (Spodoptera exigua) and specialist (Trirhabda virgata) leaf-feeding insects. Insects were reared on detached leaves (Spodoptera) or potted host plants (Trirhabda) and mortality and mass were measured. Trirhabda larvae showed little variation in survival or pupal mass attributable to either cytotype or plant origin. Spodoptera larvae were more sensitive to both cytotype and plant origin: they grew best on European tetraploids and poorly on US diploids (high mortality) and US tetraploids (low larval mass). These results show that both cytotype and plant origin influence insect herbivores, but that generalist and specialist insects may respond differently.Key words: polyploidy, cytotype, Solidago gigantea, insect herbivore, herbivory, invasive plant, introduced plantPolyploidy, or the possession of more than two sets of homologous chromosomes, is a fundamental force in angiosperm evolution.^1,2 Many plant species or species complexes consist of multiple cytotypes that may occur sympatrically;³ this is an important source of genetic structure in plant populations that is often overlooked.⁴ Possession of multiple genomes may confer advantages to polyploid plants such as increased heterozygosity, a decreased probability of inbreeding depression, or a greater gene pool available for selection; these traits contribute to the widespread success of polyploids and may make them prone to invasiveness.^5,6 In a recent article,⁷ we examined the functional consequences of polyploidy for different cytotypes of Solidago gigantea Ait. (Asteraceae), collected from both its native range (North America) and its introduced range (Europe). In this addendum, we show how cytotype and continent of origin influence interactions of S. gigantea with insect herbivores. Interactions with herbivores are expected to vary with cytotype because of phenotypic changes associated with polyploidy, but this area has received little study (reviewed in refs. ^8–11). Plant origin, from either the native range or an introduced range, should also influence herbivores. Plants may escape from their specialist natural enemies in the introduced range, thereby experiencing reduced herbivore pressure from an insect community dominated by generalists.^12,13 Given sufficient time, plants from the introduced range may evolve to decrease investment in anti-herbivore defenses, particularly those effective against specialists.¹⁴ While a growing body of research has addressed whether plant defenses against herbivory are lower in the introduced range,^12,15,16 few of these studies have also examined the influence of cytotype.¹⁷Three cytotypes of S. gigantea can be found in its native range in North America (diploid, tetraploid and hexaploid, 2n = 18, 36 and 54 respectively). These are morphologically indistinguishable and not generally treated as separate species.¹⁸ In Europe, where S. gigantea was introduced in the mid 18^th century,¹⁹ tetraploids are the dominant cytotype but diploids also occur. S. gigantea supports a diverse array of insect herbivores in its native range, but has few natural enemies in its introduced range.²⁰ We report here on experiments using both a generalist and a specialist leaf-chewing insect. The generalist, Spodoptera exigua (Lepidoptera: Noctuidae) is widely distributed and highly polyphagous, while the specialist Trirhabda virgata (Coleoptera: Chrysomelidae) feeds only on closely-related species within the genus Solidago. T. virgata is an outbreak insect that can be a major defoliator of S. gigantea and related species in North America.²¹ We grew plants originating from 10 populations in the US and 20 populations in Europe in common gardens at the University of Wisconsin-Milwaukee Field Station in Saukville, Wisconsin. There were five plant origin-cytotype combinations: three cytotypes from the US and two from Europe. Insects were reared on detached leaves from a single plant (Spodoptera) or on potted host plants (Trirhabda), for a set period of 21 d (Spodoptera) or until pupation (Trirhabda). We recorded insect survival and mass at the end of 21 d (Spodoptera) or at pupation (Trirhabda) (reviewed in ref. ²²).Overall survival was much better for the specialist Trirhabda than for the generalist Spodoptera (91% vs. 72%). Spodoptera larvae are not generally found on S. gigantea in the field, and while they are able to complete development, we found that this plant was not an ideal host. Spodoptera larvae were more sensitive to differences among cytotype and plant origin than were Trirhabda larvae. Percent survival was particularly poor for Spodoptera larvae reared on diploids from the US, where slightly more than half of the caterpillars survived for 21 days (Fig. 1). Trirhabda pupal mass was remarkably consistent across the five ploidy-plant origin combinations. In contrast, Spodoptera larvae responded to both cytotype and continent of origin. Surviving Spodoptera larvae did particularly well on tetraploid plants from the introduced range (Europe), and particularly poorly on tetraploids from the US (Fig. 1). We have previously reported that Spodoptera grow better on plants from Europe;²² our current results reveal that this difference is due exclusively to better growth on tetraploid plants. However, our results also show that both diploids and tetraploids from the US were poor hosts for Spodoptera: diploids because they caused high mortality and tetraploids because they resulted in poor growth. These results indicate that plants from the introduced range have reduced defenses against herbivores, even when accounting for polyploidy.Open in a separate windowFigure 1Mass ± se of S. exigua (A) and T. virgata (B) larvae reared on host plants of different cytotypes of Solidago gigantea originating from the US (native range) or europe (introduced range). Means in A followed by different letters are significantly different at p < 0.05 (ANOVA followed by multiple Student''s t-tests with Bonferroni correction). There were no significant differences in (B). Sample sizes for (A and B) shown in SpodopteraTrirhabdaNo. SurvivingInitial No.% SurvivalNo. SurvivingInitial No.% SurvivalUS-Diploid213954373995US-Tetraploid709375829289US-Hexaploid162467232496EU-Diploid152365232496EU-Tetraploid1011297811412988Open in a separate windowInsects were reared on a single genotype of each cytotype-origin combination for 21 days (Spodoptera) or until pupation (Trirhabda). Sample sizes for each cytotype-origin combination vary because cytotypes were not known at the time plants were collected; these distributions represent frequencies of cytotypes in our collections.Effects of the host plant on Spodoptera were probably driven, at least in part, by changes in secondary chemistry. We have previously shown that foliar terpenoids, chemicals known to influence insect herbivores,^23,24 are affected by both cytotype and continent of origin.⁷ It is surprising that Trirhabda larvae were not more sensitive to these differences in secondary chemistry among the five ploidy-origin combinations, given that Trirhabda is known to respond to host-plant chemistry.²³ We have previously reported that Trirhabda growth does not differ on European and US plants²² and show here that accounting for cytotype does not change this conclusion. In a recent study on the closely-related Solidago altissima, Halverson et al.¹¹ reported that the effects of plant cytotype on 5 gall-making herbivores were complex and not easily characterized. All five herbivores responded to plant cytotype, but for four of the five insects the most preferred cytotype was not consistent across sites. It is possible in our study that Trirhabda were responding to cytotype at a finer scale than that examined here. There may be differences due to cytotype that shift among the populations that we sampled, and that are averaged out when examined at the continental scale. We lack sufficient replication of cytotypes within populations to test this possibility. Even so, our results reported here reveal that plant cytotype can be an important source of variation affecting insect herbivores, but that generalist and specialist insects may respond differently.     

Chitosan as a MAMP,searching for a PRR

Chitosan, a deacetylated chitin derivative, behaves like a general elicitor, inducing a non-host resistance and priming a systemic acquired immunity. The defence responses elicited by chitosan include rising of cytosolic H⁺ and Ca²⁺, activation of MAP-kinases, callose apposition, oxidative burst, hypersensitive response (HR), synthesis of abscissic acid (ABA), jasmonate, phytoalexins and pathogenesis related (PR) proteins. Putative receptors for chitosan are a chitosan-binding protein, recently isolated, and possibly the chitin elicitor-binding protein (CEBiP). Nevertheless, it must be pointed out that biological activity of chitosan, besides the plant model, strictly depends on its physicochemical properties (deacetylation degree, molecular weight and viscosity), and that there is a threshold for chitosan concentration able to switch the induction of a cell death programme into necrotic cell death (cytotoxicity).Key words: chitosan, induced resistance, MAMP, PAMP, PCD, PRR, SARRecognition of microbe-associated molecular patterns (MAMPs), by pattern recognition receptors (PRRs), represents the major trait of innate immunity common to plants and animals. In plant immunity, MAMPs, more commonly known as general elicitors, include lipopolysaccharides (LPS), peptidoglycans, flagellin and fungal cell wall fragments (chitin/chitosan oligomers), phospholipids, oxylipins, fatty acids, sterols, proteins, double stranded RNA and methylated DNA, able to elicit a host defence response by binding to specific PRRs. In this view, chitosan, a deacetylated chitin derivative, behaves like a general elicitor, inducing a non-host resistance, by a PRR-mediated recognition, and priming a systemic acquired immunity (or systemic acquired resistance, SAR).¹ The defence responses elicited by chitosan include: raising of cytosolic Ca²⁺, activation of MAP-kinases, callose apposition, oxidative burst, hypersensitive response (HR), synthesis of abscissic acid (ABA), jasmonate, phytoalexins and pathogenesis related proteins (PR) (Fig. 1 and 2^–20Open in a separate windowFigure 1Different responses induced on Phaseolus vulgaris leaves by treatment with solutions of chitosan with 85% deacetylation degree and different molecular weights; all solutions have been prepared at 0.15% w/v in 0.05 M acetic acid and adjusted at pH 5.6. Callose detection with aniline blue (A–C) 12 h after treatment shows that 76 kD-chitosan elicits the formation of a network of small bright yellow fluorescent spots (B) due to callose apposition between the plasmalemma and the cell wall of some mesophyll cells (see the enlargement in the inset), while 6 kD-chitosan induces lesions involving numerous cells fluorescing in yellow-orange, possibly as consequence of the overlap of phenolics autofluorescence and callose fluorescence (see the enlargement in the inset). In 322 kD chitosan-treated leaves numerous green-fluorescent patches (C), due to chitosan deposits, are present on the leaf epidermis along cell walls, but rarely callose apposition is present. Detection of H₂O₂ deposits (as brownish precipitates in D–F) with 3-3′-diaminobenzydine (DAB), 24 h after 6 kD-chitosan treatment, indicates that the lesions in (A) are constituted by necrotizing cells as a consequence of extensive H₂O₂ deposition (D). At the same time, leaves treated with 76 kD-chitosan show moderate H₂O₂ deposits limited to the same mesophyll cells involved in callose apposition, and often localized around the substomatal cavity into which chitosan can permeate (E, arrow). No H₂O₂ deposition is present in 322 kD-chitosan treated plants (F). Evans blue staining to detect dead cells (stained in blue) 24 h after treatment (G–I) shows that the lesions in (A) have already evolved in extensive necrotic cell death, while only some of the cells with callose deposition visible in (B) had turned to programmed cell death (H) (as previously shown with other techniques). No dead cells are present in leaves treated with 322 kD-chitosan (I).

Table 1

Defence responses elicited by chitosan

Allelic frequency and genotypes of prion protein at codon 136 and 171 in Iranian Ghezel sheep breeds

PrP genotypes at codons 136 and 171 in 120 Iranian Ghezel sheep breeds were studied using allele-specific PCR amplification and compared with the well-known sheep breeds in North America, the United States and Europe. The frequency of V allele and VV genotype at codon 136 of Ghezel sheep breed was significantly lower than AA and AV. At codon 171, the frequency of allele H was significantly lower than Q and R. Despite the similarities of PrP genotypes at codons 136 and 171 between Iranian Ghezel sheep breeds and some of the studied breeds, significant differences were found with others. Planning of effective breeding control and successful eradication of susceptible genotypes in Iranian Ghezel sheep breeds will not be possible unless the susceptibility of various genotypes in Ghezel sheep breeds to natural or experimental scrapie has been elucidated.Key words: scrapie, Ghezel sheep breed, PrP genotyping, allele specific amplification, codon 136, codon 171Scrapie was first described in England in 1732,¹ and it is an infectious neurodegenerative fatal disease of sheep and goats belonging to the group of transmissible subacute spongiform encephalopathies (TSEs), along with bovine spongiform encephalopathy (BSE), chronic wasting disease and Creutzfeldt-Jakob disease.^2,3 The term prion, proteinaceous infectious particles, coined by Stanley B. Prusiner, was introduced, and he presents the idea that the causal agent is a protein.⁴ Prion proteins are discovered in two forms, the wild-type form (PrP^c) and the mutant form (PrP^Sc).⁵ Although scrapie is an infectious disease, the susceptibility of sheep is influenced by genotypes of the prion protein (PrP) gene.^2,6 Researchers have found that the PrP allelic variant alanine/arginine/arginine (ARR) at codons 136, 154 and 171 is associated with resistance to scrapie in several breeds.^7–14 Most of the sheep populations in the Near East and North African Region (84% of the total population of 255 million) are raised in Iran, Turkey, Pakistan, Sudan, Algeria, Morocco, Afghanistan, Syria and Somalia.¹⁵ In 2003, the Iranian sheep population was estimated at 54,000,000 head. The Ghezel sheep breed, which also is known as Kizil-Karaman, Mor-Karaman, Dugli, Erzurum, Chacra, Chagra, Chakra, Gesel, Gezel, Kazil, Khezel, Khizel, Kizil, Qezel, Qizil and Turkish Brown, originated in northwestern Iran and northeastern Turkey. By considering sheep breeds as one of the main sources of meat, dairy products and related products, a global screening attempt is started in different areas. In compliance with European Union Decision 2003/100/EC, each member state has introduced a breeding program to select for resistance to TSEs in sheep populations to increase the frequency of the ARR allele. A similar breeding program is established in United States and Canada. The Near East and North African Region still needs additional programs to help the global plan of eradication of scrapie-susceptible genotypes. The current study was the first to assess the geographical and molecular variation of codons 136 and 171 polymorphism between Iranian Ghezel sheep breed and well-known sheep breeds.Polymorphism at codon 136 is associated with susceptibility to scrapie in both experimental and natural models.^10,11,13,16 17 and Austrian Carynthian sheep.¹⁸ Swiss White Alpine showed higher frequency of allele V at position 136 than Swiss Oxford Down, Swiss Black-Brown Mountain and Valais Blacknose.¹⁹ Comparison of polymorphism at codon 136 in the current study with some of other breeds (20 some flock of Hampshire sheep²¹ with current study, but the frequency of it is higher than that of some other breeds.

Table 1

Comparison of PrP allelic and genotype frequencies at codon 136 in different breeds