Local plant responses to global problems: Dactylis glomerata responses to different traffic pollutants on roadsides.

The growing number of road vehicles is a major source of regional and global atmospheric pollution increasing concentrations of CO2 in the air, and levels of metals in air and soil. Nevertheless, the effects of these pollutants on plants growing at roadsides are poorly documented. We carried out an observational study of unmanipulated plants growing by the road, to identify the morpho-physiological responses in a perennial grass Dactylis glomerata. Firstly, we wanted to know the general effect of traffic intensity and ambient CO2 and its interactions on different plant traits. Accordingly, we analyzed the photosynthetic response by field A/Ci Response Curves, SLA, pigment pools, foliar nitrogen, carbohydrates and morphological traits in plants at three distances to the road. Secondly, we wanted to know if Dactylis glomerata plants can accumulate metals present on the roadside (Pb, Zn, Cu, and Sr) in their tissues and rhizosphere, and the effect of these metals on morphological traits. The MANCOVA whole model results shown: 1) a significant effect of road ambient CO2 concentration on morphological traits (not affected by traffic intensity, P interaction CO2 x traffic intensity>0.05), that was mainly driven by a significant negative relationship between the inflorescence number and ambient CO2; 2) a positive and significant relationship between ambient CO2 and the starch content in leaves (unaffected by traffic intensity); 3) a reduction in Jmax (electron transport rate) at high traffic intensity. These lines of evidences suggest a decreased photosynthetic capacity due to high traffic intensity and high levels of ambient CO2. In addition, Pb, Cu, Zn and Sr were detected in Dactylis glomerata tissues, and Cu accumulated in roots. Finally, we observed that Dactylis glomerata individuals growing at the roadside under high levels of CO2 and in the presence of metal pollutants, reduced their production of inflorescences.

Temporal matching among diurnal photosynthetic patterns within the crown of the evergreen sclerophyll Olea europaea L.

Trees are modular organisms that adjust their within-crown morphology and physiology in response to within-crown light gradients. However, whether within-plant variation represents a strategy for optimizing light absorption has not been formally tested. We investigated the arrangement of the photosynthetic surface throughout one day and its effects on the photosynthetic process, at the most exposed and most sheltered crown layers of a wild olive tree (Olea europaea L.). Similar measurements were made for cuttings taken from this individual and grown in a greenhouse at contrasted irradiance-levels (100 and 20% full sunlight). Diurnal variations in light interception, carbon fixation and carbohydrate accumulation in sun leaves were negatively correlated with those in shade leaves under field conditions when light intensity was not limiting. Despite genetic identity, these complementary patterns were not found in plants grown in the greenhouse. The temporal disparity among crown positions derived from specialization of the photosynthetic behaviour at different functional and spatial scales: architectural structure (crown level) and carbon budget (leaf level). Our results suggest that the profitability of producing a new module may not only respond to construction costs or light availability, but also rely on its spatio-temporal integration within the productive processes at the whole-crown level.

Contrasting patterns of morphological and physiological differentiation across insular environments: phenotypic variation and heritability of light-related traits in Olea europaea.

Phenotypic variation of traits can reflect the ability of plants to adjust to particular environments, but how much of this variation is heritable is not frequently analyzed in natural populations. In the present paper, we investigated the patterns of phenotypic expression in light-related leaf traits of Olea europaea subsp. guanchica, a woody sclerophyllous species endemic to the Canary Islands. We explored phenotypic differentiation and heritable variation across several island populations differing in light environment. A suite of morpho-functional (leaf size, SLA and leaf angle) and physiological (pigment pools: Chl a/b ratio, xantophyll cycle and ß-carotene) traits was measured in six populations on three islands. In addition, we estimated heritabilities for these traits following Ritland's method. Variation in morpho-functional, but not in physiological, traits was observed across the islands and was significantly related to the amount of diffuse light experienced by each population. In addition, significant heritabilities were found for morpho-functional traits, whereas expression of similar phenotypes among populations was accompanied by a lack of heritable variation in physiological traits. Most recently established populations did not exhibit lower heritabilities in quantitative traits than older populations, and apparently displayed congruent phenotypes under the local conditions. Our results strongly support the idea that different types of traits show contrasted levels of genetic and phenotypic variation in populations experiencing marked environmental differences.

Variation in sclerophylly among Iberian populations of Quercus coccifera L. is associated with genetic differentiation across contrasting environments.

Evergreen oaks are an emblematic element of the Mediterranean vegetation and have a leaf phenotype that seems to have remained unchanged since the Miocene. We hypothesise that variation of the sclerophyll phenotype among Iberian populations of Quercus coccifera is partly due to an ulterior process of ecotypic differentiation. We analysed the genetic structure of nine Iberian populations using ISSR fingerprints, and their leaf phenotypes using mean and intracanopy plasticity values of eight morphological (leaf angle, area, spinescence, lobation and specific area) and biochemical traits (VAZ pool, chlorophyll and beta-carotene content). Climate and soil were also characterised at the population sites. Significant genetic and phenotypic differences were found among populations and between NE Iberia and the rest of the populations of the peninsula. Mean phenotypes showed a strong and independent correlation with both genetic and geographic distances. Northeastern plants were smaller, less plastic, with smaller, spinier and thicker leaves, a phenotype consistent with the stressful conditions that prevailed in the steppe environments of the refugia within this geographic area during glaciations. These genetic, phenotypic, geographic and environmental patterns are consistent with previously reported palaeoecological and common evidence. Such consistency leads us to conclude that there has been a Quaternary divergence within the sclerophyllous syndrome that was at least partially driven by ecological factors.

Genetic diversity and differentiation processes in the ploidy series of Olea europaea L.: a multiscale approach from subspecies to insular populations.

Geographical isolation and polyploidization are central concepts in plant evolution. The hierarchical organization of archipelagos in this study provides a framework for testing the evolutionary consequences for polyploid taxa and populations occurring in isolation. Using amplified fragment length polymorphism and simple sequence repeat markers, we determined the genetic diversity and differentiation patterns at three levels of geographical isolation in Olea europaea: mainland-archipelagos, islands within an archipelago, and populations within an island. At the subspecies scale, the hexaploid ssp. maroccana (southwest Morocco) exhibited higher genetic diversity than the insular counterparts. In contrast, the tetraploid ssp. cerasiformis (Madeira) displayed values similar to those obtained for the diploid ssp. guanchica (Canary Islands). Geographical isolation was associated with a high genetic differentiation at this scale. In the Canarian archipelago, the stepping-stone model of differentiation suggested in a previous study was partially supported. Within the western lineage, an east-to-west differentiation pattern was confirmed. Conversely, the easternmost populations were more related to the mainland ssp. europaea than to the western guanchica lineage. Genetic diversity across the Canarian archipelago was significantly correlated with the date of the last volcanic activity in the area/island where each population occurs. At the island scale, this pattern was not confirmed in older islands (Tenerife and Madeira), where populations were genetically homogeneous. In contrast, founder effects resulted in low genetic diversity and marked genetic differentiation among populations of the youngest island, La Palma.

Winter photoinhibition in the field involves different processes in four co-occurring Mediterranean tree species.

Photoinhibition was examined in four co-occurring Mediterranean evergreen tree species during two consecutive winters. In response to low temperatures and saturating light, Juniperus phoenicea L., Pinus halepensis Mill., Quercus coccifera L. and Q. ilex ssp. ballota (Desf.) Samp. exhibited marked chronic photoinhibition, indicated by low predawn maximal photochemical efficiency of photosystem II (PSII) (Fv/Fm). Low Fv/Fm values were correlated with high concentrations of xanthophyll cycle components (VAZ) and with the maintenance of high concentrations of zeaxanthin overnight (DPSpd). In all species, however, chronic photoinhibition was enhanced as the winter progressed in the absence of changes in DPSpd, suggesting cumulative damage toward the end of winter. Photoinhibition differed among species: P. halepensis always displayed significantly higher Fv/Fm values; and Q. coccifera had the lowest Fv/Fm values, showing a high sensitivity to the combination of high light and low temperatures. Differences among species were not fully explained by differences in the xanthophyll pool or its de-epoxidation state. Chronic photoinhibition overlapped with a dynamic photoinhibition as shown by the low values of photochemical efficiency of the open reaction centers of PSII at midday. Winter photoprotective strategies differed among species and may involve photoprotective mechanisms in addition to those associated with xanthophylls. The observed species-specific differences matched results obtained for the same species in summer; however, comparison of the two seasons suggests that the higher VAZ concentration observed in winter has an additional structural photoprotective role.

Determination of L-ascorbic acid in Lycopersicon fruits by capillary zone electrophoresis.

This study shows an improved method for the determination of L-ascorbic acid (l-AA) in fruits of Lycopersicon by capillary zone electrophoresis (CZE). Two backgrounds electrolytes (BGEs) have been tested: (i) 400 mM borate at pH 8.0 and 1 x 10(-2)% hexadimethrine bromide, for the separation of Eulycopersicon subgenus species; and (ii) as in BGE(i) but supplemented with 20% (v/v) acetonitrile, for the separation of species of the Eriopersicon subgenus. The present procedures were compared with two routine methods-enzymatic assay and potentiometric titration with 2,6-dichlorophenol-indophenol. While these routine methods presented some difficulties in quantifying l-AA in several Lycopersicon fruits, CZE was successfully applied in all the analyzed samples. The proposed CZE protocols give lower detection limits (<0.4 microg ml(-1)); are cheaper, quicker, and highly reproducible; and can be applied to analyze large series of samples (ca. 50 samples per day) which is utmost importance, not only in screening trials for internal quality and tomato breeding programs, but also in systematic and routine characterization of Lycopersicon fruits.

Energy dissipation in drought-avoiding and drought-tolerant tree species at midday during the Mediterranean summer.

Photosynthetic performance was monitored during two consecutive summers in four co-occurring evergreen Mediterranean tree species growing on a south-facing rocky slope. In response to midday water stress, the drought-avoiding species Pinus halepensis Mill. exhibited marked stomatal closure (g(s)) but no changes in stem water potential (Psi(s)), whereas the drought-tolerant species Quercus coccifera L., Q. ilex ssp. ballota (Desf.) Samp. and Juniperus phoenicea L. displayed declines in midday g(s) and Psi(s). The higher resistance to CO(2) influx in needles of P. halepensis compared with the other species did not result in either a proportional increase in non-radiative dissipation of excess energy or photo-inactivation of photosystem II (PSII). No significant differences were found among species either in the de-epoxidation state of the xanthophyll cycle (DPS) or in the pool of its components on a total chlorophyll basis (VAZ). Despite contrasting midday assimilation rates, the three drought-tolerant species all exhibited a pronounced drop in photochemical efficiency at midday that was characterized by a decrease in the excitation capture efficiency of the open PSII centers. Although photoinhibition was not fully reversed before dawn, it apparently did not result in cumulative photo-damage. Thus, the drought-avoiding and drought-tolerant species employed different mechanisms for coping with excess light during the midday depression in photosynthesis that involved contrasting midday photochemical efficiencies of PSII and different degrees of dynamic photoinhibition as a photo-protective mechanism. These behaviors may be related to the different mechanisms employed by drought-avoiding and drought-tolerant species to withstand water deficit.

Long-term responses of the green-algal lichen Parmelia caperata to natural CO2 enrichment.

Acclimation to elevated CO2 was investigated in Parmelia caperata originating from the vicinity of a natural CO2 spring, where the average daytime CO2 concentration was 729 ± 39 µmol mol-1 dry air. Thalli showed no evidence of a down-regulation in photosynthetic capacity following long-term exposure to CO2 enrichment in the field; carboxylation efficiency, total Ribulose bisphosphate carboxylase/oxygenase (Rubisco) content, apparent quantum yield of CO2 assimilation, and the light-saturated rate of CO2 assimilation (measured under ambient and saturating CO2 concentrations) were similar in thalli from the naturally CO2 enriched site and an adjacent control site where the average long-term CO2 concentration was about 355 µmol mol-1. Thalli from both CO2 environments exhibited low CO2 compensation points and early saturation of CO2 uptake kinetics in response to increasing external CO2 concentrations, suggesting the presence of an active carbon-concentrating mechanism. Consistent with the lack of significant effects on photosynthetic metabolism, no changes were found in the nitrogen content of thalli following prolonged exposure to elevated CO2. Detailed intrathalline analysis revealed a decreased investment of nitrogen in Rubisco in the pyrenoid of algae located in the elongation zone of thalli originating from elevated CO2, an effect associated with a reduction in the percentage of the cell volume occupied by lipid bodies and starch grains. Although these differences did not affect the photosynthetic capacity of thalli, there was evidence of enhanced limitations to CO2 assimilation in lichens originating from the CO2-enriched site. The light-saturated rate of CO2 assimilation measured at the average growth CO2 concentration was found to be significantly lower in thalli originating from a CO2-enriched atmosphere compared with that of thalli originating and measured at ambient CO2. At lower photosynthetic photon flux densities, the light compensation point of net CO2 assimilation was significantly higher in thalli originating from elevated CO2, and this effect was associated with higher usnic acid content.

Prevalence and characteristics of enteropathogenic Escherichia coli with the eae gene in diarrhoeic rabbits.

A field study was carried out with the objective of investigating the prevalence of enteropathogenic Escherichia coli (EPEC) with the eae gene in diarrhoeic rabbits. EPEC eae+ were isolated from 60 (74%) of 81 diarrhoeic rabbits sampled in 30 industrial fattening farms localized in the four provinces of Galicia (northwestern Spain). Attaching and effacing lesions were found in 44 of 50 animals processed for histology. The 111 E. coli strains identified belonged to 19 different O serogroups and 13 biotypes. However, 53 (48%) of the strains belonged to serogroup O103 and 36 (32%) showed the serobiotype O103:B14. The eae gene was significantly more frequent (100%; 47 of 47) among the highly pathogenic rhamnose-negative strains of serobiotypes O103:B6 and O103:B14 than among the E. coli strains belonging to other serobiotypes (36%; 23 of 64) (P < 0.001). In this first report about the prevalence of EPEC with the eae gene in rabbits, we conclude that the class of E. coli strains observed is a common cause of diarrhoea in Galician rabbit farms, and that highly pathogenic rhamnose-negative strains of serotype O103:K-:H2 and biotype B14 are specially predominant.

O serogroups, biotypes, and eae genes in Escherichia coli strains isolated from diarrheic and healthy rabbits.

A total of 305 Escherichia coli strains isolated from diarrheic and healthy rabbits in 10 industrial fattening farms from different areas of Spain were serotyped, biotyped, and tested for the presence of the eae gene and toxin production. The characteristics found in strains isolated from healthy rabbits were generally different from those observed in E. coli strains associated with disease. Thus, strains with the eae gene (74% versus 22%); strains belonging to serogroups O26, O49, O92, O103, and O128 (64% versus 12%); rhamnose-negative strains (51% versus 5%); and rhamnose-negative O103 strains with eae genes present (41% versus 1%) were significantly (P < 0.001 in all cases) more frequently detected in isolates from diarrheic animals than in those from healthy rabbits. Whereas a total of 35 serogroups and 17 biotypes were distinguished, the majority of the strains obtained from diarrheic rabbits belonged to only four serobiotypes, which in order of frequency were O103:B14 (72 strains), O103:B6 (16 strains), O26:B13 (12 strains), and O128:B30 (12 strains). These four serobiotypes accounted for 48% (112 of 231) and 5% (4 of 74) of the E. coli strains isolated from diarrheic and healthy rabbits, respectively. Only six strains were toxigenic (three CNF1+, two CNF2+, and one VT1+). We conclude that enteropathogenic E. coli strains that possess the eae gene are a common cause of diarrhea in Spanish rabbit farms and that the rhamnose-negative highly pathogenic strains of serotype O103:K-:H2 and biotype B14 are especially predominant. Detection of the eae gene is a useful method for the identification of enteropathogenic E. coli strains from rabbits. However, a combination of serogrouping and biotyping may be sufficient to accurately identify the highly pathogenic strains for rabbits.

A poorly-differentiated squamous cell thymoma in a chicken with lymphoma.

Thymic carcinomas are rare in birds. A poorly-differentiated squamous cell thymoma was found in a chicken in association with a lymphoma caused by Marek's disease. Immunohistochemical techniques with different tumour markers were applied in order to determine the histogenesis of the thymic tumour cells. A positive immunoreaction of the tumour cells to cytokeratin, considered a specific marker for epithelial tumours, confirmed the diagnosis of a thymoma.

The projections to the medulla of neurons innervating the carotid sinus in the dog.

The localization and brain stem projections of neurons innervating the carotid sinus of the dog were studied by horseradish peroxidase histochemistry following microinjection of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) under the adventitia of the carotid sinus. Within the brain stem, labeled afferent fibers and presumptive terminals were found bilaterally in the caudal nucleus tractus solitarius (nTS), the area postrema (AP), and the lateral tegmental field (LTF), reaching the area of the nucleus ambiguus (nA). Sparse labeling was also seen in the ipsilateral spinal trigeminal nucleus (SpV) and lateral cuneatus nucleus (LCn). These findings suggest the existence of multiple pathways by which peripheral baroreceptor inputs may influence central cardiovascular-related neurons. In addition to classically defined relay in the nTS, carotid sinus afferents may also interact more directly with these neurons in other brain stem regions.

The localization of neurons innervating the carotid sinus in the dog.

The localization of neurons innervating the carotid sinus of the dog was studied by horseradish peroxidase histochemistry following microinjection of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) under the adventitia of the carotid sinus. Labeled cell bodies were found in the petrosal (198 +/- 108; mean +/- S.D.) and superior cervical ganglia (SCG) (890 +/- 354 mean +/- S.D.) supporting the existence of both a sensory afferent and a sympathetic efferent innervation of the carotid sinus. Labeled neurons in the petrosal ganglion were round pseudounipolar neurons of variable size. Labeled neurons in the SCG were multipolar and appeared distributed over the whole ganglion, but with a higher density toward its caudal half. No labeled perikarya appeared either in the brainstem or in the nodose or jugular ganglia, suggesting that in the dog a vagal pathway for carotid sinus baroreceptor afferents does not exist.

Prolactinoma in a sheep.

This report describes the incidental finding of an acidophilic adenoma of the pituitary gland in an adult ewe with clinical signs of a nervous disorder. Immunohistochemical examination demonstrated prolactin in the tumour cells, justifying the use of the term "prolactinoma" for the first time in veterinary medicine. This tumour should be considered in the differential diagnosis of nervous diseases of sheep.

Immunoreactivity to chromogranin and to vasoactive intestinal peptide in a canine phaeochromocytoma.

A canine phaeochromocytoma was diagnosed by (1) argyrophil methods, (2) immunoreactivity to chromogranin and to vasoactive intestinal peptide (VIP), and (3) the demonstration of typical electron-dense granules by transmission electron microscopy. This is the first report of immunoreactivity to general neuroendocrine markers such as chromogranin, and to VIP, in a phaeochromocytoma in domestic animals. The use of these markers for the differential diagnosis of phaeochromocytoma, and the role of VIP in the severe chronic diarrhoea shown by the dog in this study are discussed.

Coexistence of serotonin and cholecystokinin in paraneurons of the foetal sheep lung.

The coexistence of serotonin and cholecystokinin was studied in foetal sheep lungs at pseudoglandular stage of development by light microscopic immunohistochemistry. The coexistence was examined by staining consecutive sections with the different antibodies. Serotonin and cholecystokinin immunoreactivity was found within consecutive sections of most bronchopulmonary neuroepithelial bodies and in consecutive sections of the same intrapulmonary autonomic ganglia.

Localization of serotonin, cholecystokinin and somatostatin immunoreactivity in the lower respiratory tract of embryonic, foetal and postnatal sheep.

The lower respiratory tract of the sheep was studied by light-microscopical immunocytochemistry for serotonin, cholecystokinin, somatostatin, bombesin and calcitonin during different periods of lung development; embryonic, foetal and postnatal. At embryonic period only intraepithelial serotonin-containing cells as solitary neuroendocrine cells (NEC) and neuroepithelial bodies (NEB) were found. At foetal stages, immunoreactive cells to serotonin, cholecystokinin and somatostatin were observed in airway epithelium, as solitary NEC and NEBs, and in autonomic intrapulmonary ganglia as single or clusters of small intensely-fluorescent (SIF) cells. In postnatal sheep, serotonin- and cholecystokinin-containing cells were found within airway mucosa as solitary NECs and NEBs. No immunoreactive cells were observed with antiserum to bombesin and calcitonin. Quantitative studies showed that serotonin was the predominant substance, and that solitary neuroendocrine cells were more numerous in distal conducting airways and at foetal stages.

Quantitative assessment of ATPase-positive epidermal dendritic (Langerhans) cells in piglets and adult swine.

To assess the distribution pattern of Langerhans cells (LC) in normal porcine skin, epidermal sheets from six anatomical sites from three age-matched groups of male and female pigs were stained for ATPase activity. This histoenzymological technique is considered specific for Langerhans cells in normal epidermis. No statistically significant differences were observed between mean Langerhans cell density per mm2 of epidermis from male and female pigs, nor between different anatomical sites in the same age group. Statistically significant differences (P less than 0.0005) were observed when comparing group A one- to two-week-old piglets (463 to 518 LC mm-2) with group B six month olds (641 to 804 LC mm-2) and group C two years and over sows (741 to 830 LC mm-2). Morphological variations in the skin of young piglets, much thinner and with rudimentary or no epidermal rete pegs, could account for this significant variation.

Ultrastructural identification of Langerhans cells in normal swine epidermis.

Langerhans cells of the epidermis of 6-month-old white crossbred farm pigs were identified by electron microscopy. Ultrastructurally they were similar to those described in other mammals. They were present in basal and suprabasal layers and were characterised by a lobulated nucleus and an electrolucent cytoplasm with occasional dendritic processes, and the absence of tonofilaments and specialised unions with surrounding keratinocytes. They were specifically identified by the presence of characteristic rod or racquet-shaped intracytoplasmic granules. Intraepidermal clear cells without specific granules were present, although no melanocytes were observed. This is the first report of the presence of Birbeck granules in porcine Langerhans cells.