Effect of plant neutrophil elastase inhibitor on leucocyte migration, adhesion and cytokine release in inflammatory conditions

BACKGROUND AND PURPOSE The serine and cysteine peptidase inhibitor, BbCI, isolated from Bauhinia bauhinioides seeds, is similar to the classical plant Kunitz inhibitor, STI, but lacks disulphide bridges and methionine residues. BbCI blocks activity of the serine peptidases, elastase (K(iapp) 5.3 nM) and cathepsin G (K(iapp) 160.0 nM), and the cysteine peptidase cathepsin L (K(iapp) 0.2 nM). These three peptidases play important roles in the inflammatory process. EXPERIMENTAL APPROACH We measured the effects of BbCI on paw oedema and on leucocyte accumulation in pleurisy, both induced by carrageenan. Leucocyte-endothelial cell interactions in scrotal microvasculature in Wistar rats were investigated using intravital microscopy. Cytokine levels in pleural exudate and serum were measured by ELISA. KEY RESULTS Pretreatment of the animals with BbCI (2.5 mg.kg(-1)), 30 min before carrageenan-induced inflammation, effectively reduced paw oedema and bradykinin release, neutrophil migration into the pleural cavity. The number of rolling, adhered and migrated leucocytes at the spermatic fascia microcirculation following carrageenan injection into the scrotum were reduced by BbCI pretreatment. Furthermore, levels of the rat chemokine cytokine-induced neutrophil chemo-attractant-1 were significantly reduced in both pleural exudates and serum from animals pretreated with BbCI. Levels of interleukin-1 beta or tumour necrosis factor-alpha, however, did not change. CONCLUSIONS AND IMPLICATIONS Taken together, our data suggest that the anti-inflammatory properties of BbCI may be useful in investigations of other pathological processes in which human neutrophil elastase, cathepsin G and cathepsin L play important roles.

Studies of aberrant phyllotaxy1 Mutants of Maize Indicate Complex Interactions between Auxin and Cytokinin Signaling in the Shoot Apical Meristem

One of the most fascinating aspects of plant morphology is the regular geometric arrangement of leaves and flowers, called phyllotaxy. The shoot apical meristem (SAM) determines these patterns, which vary depending on species and developmental stage. Auxin acts as an instructive signal in leaf initiation, and its transport has been implicated in phyllotaxy regulation in Arabidopsis (Arabidopsis thaliana). Altered phyllotactic patterns are observed in a maize (Zea mays) mutant, aberrant phyllotaxy1 (abph1, also known as abphyl1), and ABPH1 encodes a cytokinin-inducible type A response regulator, suggesting that cytokinin signals are also involved in the mechanism by which phyllotactic patterns are established. Therefore, we investigated the interaction between auxin and cytokinin signaling in phyllotaxy. Treatment of maize shoots with a polar auxin transport inhibitor, 1-naphthylphthalamic acid, strongly reduced ABPH1 expression, suggesting that auxin or its polar transport is required for ABPH1 expression. Immunolocalization of the PINFORMED1 (PIN1) polar auxin transporter revealed that PIN1 expression marks leaf primordia in maize, similarly to Arabidopsis. Interestingly, maize PIN1 expression at the incipient leaf primordium was greatly reduced in abph1 mutants. Consistently, auxin levels were reduced in abph1, and the maize PIN1 homolog was induced not only by auxin but also by cytokinin treatments. Our results indicate distinct roles for ABPH1 as a negative regulator of SAM size and a positive regulator of PIN1 expression. These studies highlight a complex interaction between auxin and cytokinin signaling in the specification of phyllotactic patterns and suggest an alternative model for the generation of altered phyllotactic patterns in abph1 mutants. We propose that reduced auxin levels and PIN1 expression in abph1 mutant SAMs delay leaf initiation, contributing to the enlarged SAM and altered phyllotaxy of these mutants.

Cell-cell signal-dependent dynamic interactions between HD-GYP and GGDEF domain proteins mediate virulence in Xanthomonas campestris

RpfG is a paradigm for a class of widespread bacterial two-component regulators with a CheY-like receiver domain attached to a histidine-aspartic acid-glycine-tyrosine-proline (HD-GYP) cyclic di-GMP phosphodiesterase domain. In the plant pathogen Xanthomonas campestris pv. campestris (Xcc), a two-component system comprising RpfG and the complex sensor kinase RpfC is implicated in sensing and responding to the diffusible signaling factor (DSF), which is essential for cell-cell signaling. RpfF is involved in synthesizing DSF, and mutations of rpfF, rpfG, or rpfC lead to a coordinate reduction in the synthesis of virulence factors such as extracellular enzymes, biofilm structure, and motility. Using yeast two-hybrid analysis and fluorescence resonance energy transfer experiments in Xcc, we show that the physical interaction of RpfG with two proteins with diguanylate cyclase (GGDEF) domains controls a subset of RpfG-regulated virulence functions. RpfG interactions were abolished by alanine substitutions of the three residues of the conserved GYP motif in the HD-GYP domain. Changing the GYP motif or deletion of the two GGDEF-domain proteins reduced Xcc motility but not the synthesis of extracellular enzymes or biofilm formation. RpfG-GGDEF interactions are dynamic and depend on DSF signaling, being reduced in the rpfF mutant but restored by DSF addition. The results are consistent with a model in which DSF signal transduction controlling motility depends on a highly regulated, dynamic interaction of proteins that influence the localized expression of cyclic di-GMP.

Morphological patterns of extrafloral nectaries in woody plant species of the Brazilian cerrado

Extrafloral nectaries are nectar-secreting structures that are especially common among the woody flora of the Brazilian cerrado, a savanna-like vegetation. In this study, we provide morphological and anatomical descriptions of extrafloral nectaries (EFNs) occurring on vegetative and reproductive organs of several plant species from the cerrado, and discuss their function and ecological relevance. We describe the morphology and anatomy of EFNs of 40 species belonging to 15 woody families using scanning electron microscopy and light microscopy. We categorise EFNs following a structural-topographical classification, and characterise the vascularised and complex nectaries, amorphous nectaries and secretory trichomes. Fabaceae, Bignoniaceae, Malpighiaceae and Vochysiaceae were the plant families with the majority of species having EFNs. Ten species possess more than one morphotype of gland structure. Observations and experimental field studies in the cerrado support the anti-herbivore role of EFN-gathering ants in this habitat. Additional morphological studies of EFNs-bearing plants, including other growth forms (e.g. herbs and lianas), are being undertaken and will hopefully cast further light on the ecological relevance of these glands in the cerrado, especially with respect to their attractiveness to multiple visitors.

Bird attributes, plant characteristics, and seed dispersal of Pera glabrata (Schott, 1858), (Euphorbiaceae) in a disturbed cerrado area

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Habitat selection and potential antiherbivore effects of Peucetia flava (Oxyopidae) on Solanum thomasiifolium (Solanaceae)

Several spider species use plants as shelter and foraging sites, but the relationships among these organisms are still poorly known. Lynx spiders of the genus Peucetia do not build webs, and many species live strictly in plants bearing glandular trichomes. Peucetia flava Keyserling 1877 inhabits Solanum thomasifolium in southeastern Brazil and usually preys on herbivores and other small insects adhered to the glandular trichomes of its host plant. To evaluate the potential anti-herbivore protection. of this spider species for S. thomasifolium, we glued termites used as herbivore models oil trichomes of S. thomasifolium and on neighboring plants lacking glandular trichomes. leaf miner damage and spider density were recorded for S. thomasifolium plants in July 1997. There was a positive relationship between plant size and spider density. The removal or termites in S. thomasifolium by P. flava was higher than ill plants without glandular trichomes. The leaf miner damage was negatively related to spider density. Our results Suggest that P. flava may be all important plant bodyguard in the defense of S. thomasifolium from its natural herbivores.

Microhabitat use by Peucetia flava (Oxyopidae) on the glandular plant Rhyncanthera dichotoma (Melastomataceae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Trait-mediated effects on flowers: Artificial spiders deceive pollinators and decrease plant fitness

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Natural history of Peucetia flava (Araneae, Oxyopidae): seasonal density fluctuation, phenology and sex ratio on the glandular plant Rhyncanthera dichotoma (Melastomataceae)

The seasonal density fluctuation, phenology and sex ratio of Peucetia flava (Oxyopidae) on Rhyncanthera dichotoma (Melastomataceae) were investigated during a year in a swamp in southeastern Brazil. Peucetia flava displayed an unusual non-seasonal life cycle and the population size varied little over the year. The density of Peucetia spiders increased with the increase in abundance of leaves and number of arthropods adhered to glandular trichomes on R. dichotoma leaves. Our findings suggest that seasonal density fluctuation of Peucetia may be influenced by foraging site availability (i.e. leaves) and prey supply (i.e. arthropods adhering to glandular trichomes). The unusual seasonal stability of P. flava may be related to the type of habitat in which this spider occurs (swamp), because of the constant input of allochthonous resources from the water source, i.e. aquatic insects that migrate to the terrestrial environment.

Interactions in the Agrobacterium-soybean system and capability of some Brazilian soybean cultivars to produce somatic embryos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

An experimental inoculation system to study citrus-Xylella fastidiosa interactions

Difficulties in reproducing the citrus variegated chlorosis (CVC) disease symptoms in expertmental plants have delayed implementation of studies to better understand the essential aspects of this important disease. In an extensive Study, cultivars of sweet orange (Citrus sinensis) were inoculated with Xylella fastidiosa using procedures that included root immersion, and stein absorption, pricking, or infiltration of the inoculum into plants of different ages. Inoculum consisted of 5-day-old cultures or cell suspensions of CVC strain 9a5c diluted in phosphate-buffered saline. Inoculated plants and controls were grown, or transferred just after inoculation, to 5-liter pots or 72-cell foam trays. Approximately 4, 5, 9, and 12 months after inoculation, leaves were collected and processed for polymerase chain reaction analysis or X. fastidiosa isolation on BCYE agar medium. Root immersion and stem inoculation of 4- and 6-month-old plants resulted in low percentages of symptomatic (0 to 7%) and plants positive by isolation (0 to 9%). Pinpricked or injected stems of I-month-old seedlings resulted in high percentages of plants symptomatic (29 and 90% in Pera Rio, 75, 59, and 83% in Valencia, and 77% in Natal) or positive by isolation (26 and 93% in Pera Rio, 98, 96, and 83% in Valencia, and 77% in Natal), In foam trays, the seedlings grew less, the incubation period was shorter. and disease severity was higher than in pots. This system allows testing of higher numbers of plants in a reduced space with a more precise reproduction of the experimental conditions.

Association of two new Coryphasia species (Araneae, Salticidae) with tank-bromeliads in southeastern Brazil: Habitats and patterns of host plant use

Two allopatric species of Coryphasia (Araneae: Salticidae), both described for the first time in this study, have been found associated with Bromeliaceae in distinct phytophysiognomies (e.g., inselbergs, highland forests and restingas) from southeastern Brazil. In this study, we investigated whether these salticids were associated specifically with bromeliads, and whether they used bromeliads of different species and sizes in distinct geographic regions. The Coryphasia species were rarely found outside bromeliads, occupied larger bromeliad species among those available, and were generally more frequent on bromeliads in open areas, such as inselbergs on mountain tops. The two Coophasia species were observed submerging in phytotelmata, possibly as an anti-predatory behavior. The patterns of spatial and microspatial distribution and the submergence behavior of these species were similar to those of other bromeliad-dwelling salticids, which suggests a convergence among spiders that live on bromeliads.

HERBIVORY AND ASSEMBLAGE STRUCTURE OF MYRMECOPHYTOUS UNDERSTORY PLANTS AND THEIR ASSOCIATED ANTS IN THE CENTRAL AMAZON

The species combinations of myrmecophytic plants were compared in three different, neighboring local central Amazon forest sites. The proportional contribution of myrmecophytes in each setting varied significantly, with Maieta guainensis being the most abundant in each locality. This pattern resulted in low site similarity values. Other recorded species were Hirtella physophora, Tachigalia myrmecophila, Duroia sp., Tococa sp., and Cordia nodosa. Little variability was found with respect to associated ants that inhabited the myrmecophytes, and mutual entropies indicated a high degree of mutualistic interactions. However, for the majority of myrmecophytes, no differences in herbivore damage levels could be attributed to the presence of ants, with only M. guianensis and T. myrmecophila demonstrating significantly lower damages when inhabited by ants. Their respective ant associates, Pheidole minitula and Pseudomyrmex concolor, were thus the only plant-ants with a demonstrable ability to reduce the levels of herbivory in their host plant.

Plant glandular trichomes mediate protective mutualism in a spider-plant system

1. Although several species of Peucetia (Oxyopidae) live strictly in association with plants bearing glandular trichomes worldwide, to date little is known about whether these associations are mutualistic.2. In this study we manipulated the presence of Peucetia flava on the glandular plant Rhynchanthera dichotoma in the rainy and post-rain season, to test the strength of its effects on leaf, bud, and flower damage and plant reproductive output. In addition, we ran independent field experiments to examine whether these sticky structures improve spider fidelity to plants.3. Peucetia suppressed some species of foliar phytophages, but not others. Although spiders have reduced levels of leaf herbivory, this phenomenon was temporally conditional, i.e. occurred only in the post-rain but not in the rainy season. Floral herbivory was also reduced in the presence of spiders, but these predators did not affect plant fitness components.4. Plants that had their glandular trichomes removed retained fewer insects than those bearing such structures. Spiders remained longer on plants with glandular trichomes than on plants in which these structures had been removed. Isotopic analyses showed that spiders that fed on live and dead labelled flies adhered to the glandular hairs in similar proportions.5. Spiders incurred no costs to the plants, but can potentially increase individual plant fitness by reducing damage to reproductive tissues. Temporal conditionality probably occurred because plant productivity exceeded herbivore consumption, thus dampening top-down effects. Specialisation to live on glandular plants may have favoured scavenging behaviour in Peucetia, possibly an adaptation to periods of food scarcity.

Ant exclusion and reproduction of Croton sarcopetalus (Euphorbiaceae)

A total of 20 insect species were observed on the extrafloral nectaries (EFNs) of Croton sarcopetalus. The most frequent ant species were Zacryptocerus sp., Crematogaster brevispinosa, C. scelerata, and Linepithema humile. Ants continuously patrolled the plants taking extrafloral nectar. Experimental data showed that there were no significant differences in either the degree of herbivory or in the reproductive output between control stems (with ants) and treated ones (without ants). We found no significant evidence of protection by ants mediated by EFNs in C. sarcopetalus. It is possible that the plant has other mechanisms than ant protection to prevent herbivore damage (e.g., hairs, latex, chemical defense). As this species occupies the southernmost distribution for the genus, the presence of EFNs in this species may be the remnant of a mutualistic interaction with the ants in tropical regions, where the abundance and diversity of ants and herbivores are comparatively higher. To draw accurate patterns in the genus Croton, further investigations in other species from tropical regions of South America are needed.