sunTILL: a TILLING resource for gene function analysis in sunflower

Background

Cultivated sunflower (Helianthus annus L.) is a globally important oilseed crop, subjected to intensive genetic and genomic studies. Although classical mutagenesis has successfully been applied to Helianthus genus in the past, we have developed the first sunflower TILLING resource.

Results

To balance the maximum mutation density with an acceptable plant survival rate, a 'kill curve' analysis was first conducted with different ethylmethanesulfonate (EMS) dosages and different exposure times. According to the germination rate, a treatment with 0.7% EMS for 6 h was chosen. An M₂ progeny of 3,651 fertile plants was obtained. Totally, 4.79% of the whole population showed clear aberrant phenotypes. A microsatellite analysis on a representative sample of the original seed stock and mutant lines confirmed the uniformity of the genetic background of plant material. The TILLING procedure was successfully applied to sunflower genome, initially by a CelI-nuclease mismatch cleavage assay coupled with a DNA-pooling level test. To investigate the efficiency of the mutagenic treatment, a pilot screening was carried out on 1,152 M₂ lines focusing on four genes, three involved in the fatty acid biosynthetic pathway and one for downy mildew resistance. A total of 9 mutant lines were identified and confirmed by sequencing; thereby, the estimated overall mutation frequency for the pilot assay resulted to be 1/475 kb.

Conclusion

A first TILLING population for a high throughput identification of EMS-induced point mutations in sunflower genome has been successfully obtained. This represents a powerful tool to a better understanding of gene function in sunflower.     

One- and two-dimensional electrophoretic identification of IgE-binding polypeptides of Lupinus albus and other legume seeds

The prevalence of food allergies in the world population requires integrated approaches to identify new potential allergens, especially those of plant origin. The aim of this work was the allergen in vitro analysis of Lupinus albus seed proteome, a promising food protein source, and the assessment of IgE cross-reactivities with other more diffused legume species. A combination of one- and two-dimensional gel electrophoresis and immunoblotting analyses with specific IgGs for band identification and lupin-sensitized patients' circulating IgEs for allergenicity studies has been used. Two lupin proteins, namely, conglutin gamma and 11S globulin basic subunits, strongly reacted with all patients' sera. Also, cross-reactivities with the homologous polypeptides of other legume species were observed. Otherwise, no reaction at all was detected with a 2S-type lupin protein. This global electrophoretic approach has allowed the identification of a new potential lupin allergen and confirmed the cross-reactivity among the legume 11S globulin basic subunits.     

Chemical characterization and antioxidant properties of coffee melanoidins

Melanoidins, the brown polymers formed through Maillard reaction during coffee roasting, constitute up to 25% of the coffee beverages' dry matter. In this study chemical characterization of melanoidins obtained from light-, medium-, and dark-roasted coffee beans, manufactured from the same starting material, was performed. Melanoidins were separated by gel filtration chromatography and studied by MALDI-TOF mass spectrometry. Results showed that the amount of melanoidins present in the brews increased as the intensity of the thermal treatment increased, while their molecular weight decreased. The antioxidant activity of melanoidins isolated from the different brews was studied by using different methodologies. Melanoidins antiradical activity determined by ABTS(*)(+) and DMPD(*)(+) assays decreased as the intensity of roasting increased, but the ability to prevent linoleic acid peroxidation was higher in the dark-roasted samples. Data suggest that melanoidins must be carefully considered when the relevance of coffee intake in human health is studied.     

Assay of aroma active components of virgin olive oils from southern Italian regions by SPME-GC/ion trap mass spectrometry

An SPME-GC/ion trap method was exploited to determine the chromatogram of volatile compounds of organic olive oils of southern Italian regions. The method is based on the assay of the terminal species of the "lipoxygenase pathway", which are present in the volatile fraction of the sampled compounds. Ethyl isobutanoate was used as internal standard in either the EI or CI ionization mode. The absolute concentration values of each analyte were evaluated through good-to-excellent calibration curves. Case studies on oils obtained from different cultivars or harvesting times are presented. The quantitative data for each compound were subjected to principal component analysis to characterize the different cultivars of this work.     

Multifocal cutaneous histiocytic sarcoma in a young dog and review of histiocytic cell immunophenotyping

A 9‐month‐old male Great Dane had progressive generalized nodular dermatopathy for several months. There were > 100 raised, alopecic, firm, painful nodules throughout the skin. Aspirates from several lesions yielded moderate numbers of irregularly round or polygonal to spindle‐shaped cells with mild to moderate anisocytosis and few inflammatory cells, and the cytologic interpretation was proliferation of mesenchymal or histiocytic cells. On histopathologic examination, nodules were composed of densely packed sheets of round to spindle‐shaped cells with mild anisokaryosis and low mitotic activity. Multifocal histiocytic sarcoma with a spindle‐cell pattern was diagnosed based on morphologic features and intense expression of CD18. Additional immunophenotypic analysis on frozen sections of tissue confirmed the diagnosis of histiocytic sarcoma; expression of CD18, CD45, CD1a, CD11b, and CD11c, limited expression of Thy‐1 (CD90) and CD80, and lack of expression of CD4, CD11d, and CD86 indicated that the cells were likely interstitial dendritic cells; a review of reactive and neoplastic dendritic cells is provided. Based on staging, internal organs were not affected. Sequential treatment with lomustine and doxorubicin failed to prevent progression of the cutaneous lesions, and the dog died 3 months after initial diagnosis. At necropsy, a focus of neoplastic cells was present in one lymph node, but except for skin other organs were not involved. The clinical presentation of histiocytic sarcoma may be unusual, and neoplastic cells may lack overt features of malignancy on cytologic and histopathologic examination. In some instances, immunophenotyping is required to differentiate histiocytic sarcoma from other histiocytic disorders.     

Application of 10% imidacloprid/50% permethrin to prevent Ehrlichia canis exposure in dogs under natural conditions

Canine monocytic ehrlichiosis (CME) caused by Ehrlichia canis is the most known canine tick-borne disease (TBD) spread throughout the world. Preventing tick bites is a priority to reduce the risk of TBDs and it was the aim of the present study to evaluate the efficacy of a combination of imidacloprid 10% and permethrin 50% (ImPer) (Advantix; Bayer AG, Germany) in a spot-on formulation to control CME under field conditions. On January-March 2005, 845 dogs from two kennels in southern Italy (kennels of Bari (KB)- and Ginosa (KG)), with a history of tick infestation were initially tested by serology and PCR assay for E. canis infection. Data on Leishmania infantum infection were also available from a previous study carried out on the same dog population. One hundred twenty-six dogs (14.9%) presented anti-E. canis antibodies with a relative prevalence of 15.6% (n=65 dogs in KB) and 14.2% (n=61 dogs in KG). Five hundred thirty-five animals found negative both for E. canis and L. infantum infections were enrolled in three groups (Group A--treated with ImPer once a month; Group B--treated every 2 weeks; and Group C--untreated control animals) and monitored for E. canis infection by serology and PCR in November 2005 (first follow-up) and in March 2006 (second follow-up). The E. canis infection was serologically revealed, at the first and/or second follow-up, in 26 animals from Group C in KB and KG (mean incidence density rate (IDR), 13.24%) while in none of the animals from Group A (KB and KG) and only in one animal from Group B (IDR 1.13%) in KG. The final protection efficacy of ImPer ranged from 95.57% to 100% in Groups B and A. At PCR only 15 dogs from KG were positive for Rickettsiales only at the first follow-up and at the sequence analysis two (both in Group C) revealed 100% homology with E. canis sequences while 13 with Anaplasma platys. Four out of 13 A. platys PCR-positive dogs were also seropositive for E. canis at one or both follow-ups. ImPer, by virtue of its repellent and acaricidal activity against ticks, has been shown to be efficacious to prevent E. canis infection in treated dogs living under natural conditions in endemic areas.     

Soil organic carbon quality in forested mineral wetlands at different mean annual temperature

Forested mineral soil wetlands (FMSW) store large stocks of soil organic carbon (SOC), but little is known on: (i) whether the quality of SOC stored in these soils (proportion of active versus more resistant SOC compounds) differs from SOC in upland soils; (ii) how the quality of SOC in FMSW varies with mean annual temperature (MAT); and (iii) whether SOC decomposition rates in these environments respond to warming and drying more strongly than those observed in upland soils. To address this substantial knowledge gap, we identified nine FMSW and fifteen paired upland forest sites across three bioregions in North America (sub-alpine in Colorado; north-temperate in Minnesota; and south-temperate in South Carolina) to test the following three hypotheses. First, FMSW store a higher proportion of active SOC compared with upland systems because long anaerobic periods favor the accumulation of labile substrates. Second, in FMSW, SOC quality decreases from cold to warm bioregions because high quality detritus accumulates preferentially at cool sites where decomposition is slow. Finally, decomposition of SOC in FMSW will respond more strongly to warming under aerobic conditions than SOC from upland forest soils because of higher accumulation of active SOC in FMSW. To test these hypotheses, we incubated FMSW and upland forest soils at two constant temperatures (10 and 30 °C) for 525-d under aerobic conditions and constant moisture. In contrast to our first hypothesis, we observed similarly rapid depletion of active SOC compounds at initial stages of incubation across FMSW and upland sites, and across the 525-d incubations we observed overall lower SOC decomposition rates in our FMSW soils. In line with our second hypothesis, and across FMWS and upland soils, we found greater SOC loss in the sub-alpine bioregion than both temperate regions. In contrast to our last hypothesis, we found no difference in the temperature sensitivity (Q₁₀) of SOC decomposition in FMSW and upland forest soils. Critically, total SOC loss (g SOC per g soil) was larger in FMSW because of the large amount of SOC stored in these ecosystems, indicating that despite a lack of difference between FMSW and upland responses, the total release of C from FMSW that could result from global warming may be large.     

Assessment of oak forest condition based on leaf biochemical variables and chlorophyll fluorescence

Pedunculate oak forests (Quercus robur L.) in the Ticino Regional Park, Italy, are declining as a result of insect attacks, summer droughts and air pollution. The assessment and monitoring of forest condition can provide a basis for managing and conserving forest ecosystems and thereby avoid loss of valuable natural resources. Currently, most forest assessments are limited to ground-based visual evaluations that are local and subjective. It is therefore difficult to compare data collected by different crews or to define reliable trends over years. We examined vegetation variables that can be quantitatively estimated by remote observations and, thus, are suitable for objective monitoring over extended forested areas. We found that total chlorophyll (Chl) concentration is the most suitable variable for assessing pedunculate oak decline. It is highly correlated with visual assessments of discoloration. Furthermore, Chl concentration can be accurately estimated from leaf optical properties, making it feasible to map Chl concentration at the canopy level from satellite and airborne remote observations.     

IgE-Mediated Cross-Reactivity among Leguminous Seed Proteins in Peanut Allergic Children

The immunological cross-reactivity among major protein- and oil-crops, including lupin, lentil, pea, peanut, kidney bean and soybean, has been studied by a combination of in vitro and in vivo experimental approaches: SDS-PAGE separations of legume protein extracts and immuno-blot revelations with 12 peanut-sensitive subjects’ sera, Immuno-CAP and Skin Prick tests on the same subjects. The immuno-blotting data showed a wide range of IgE-binding responses both displayed by one subject towards different plant extracts and among subjects. Differences were both quantitative and qualitative. The prevalent responses of most subjects’ sera were seen with peanut polypeptides, as expected, as well as with various polypeptides of the other legumes, the most recurrent of which were the basic subunits of the 11S globulins. The distribution of in vivo responses generally paralleled those obtained by in vitro approaches with strong responses elicited by peanut, lentil and pea protein extracts, especially by most sensitive subjects, thus providing a consistent overall set of results. In this work, the comparison of various approaches has allowed us to get an overall broad picture of the immunological cross-reactivities among proteins of widely used different seed species and to hypothesize the role of most conserved specific polypeptides.