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.     

Phytotoxic polyketides produced by <Emphasis Type="Italic">Phomopsis foeniculi</Emphasis>, a strain isolated from diseased Bulgarian fennel

Recently, a new fungal disease caused by Diaporthe angelicae (anamorph Phomopsis foeniculi) has been found with increasingly frequency on fennel (Foeniculum vulgare) in Bulgaria. Using a bioassay-guided isolation and purification procedure, different metabolites were isolated from the fungal culture filtrates. They were identified by spectroscopic methods as nectriapyrone, a pentaketide monoterpenoid, and altersolanols A and J and macrosporin, three octaketides anthracenones. Leaf puncture bioassay was applied on detached tomato leaves to prove the phytotoxic activity of the fractions and of pure compounds. Nectriapyrone and altersolanols A and J showed a modulated phytotoxicity, while macrosporin was not toxic. Altersolanol A was the most active compound.     

Partial replacement of Fe(o,o‐EDDHA) by humic substances for Fe nutrition and fruit quality of citrus

The most widely used Iron (Fe) fertilizer in calcareous soils is the synthetic chelate Fe(o,o‐EDDHA). However, humic substances are occasionally combined with Fe chelates in drip irrigation systems in order to lower costs. We investigated the effect of various mixtures of Fe(o,o‐EDDHA) and a commercially available humic substance on Fe availability in a calcareous soil from Murcia, Spain (in vitro experiment) and on leaf Fe content and fruit‐quality attributes of Citrus macrophylla (field experiment). In the in vitro experiment, a calcareous soil was incubated for 15 d with solutions of sole Fe(o,o‐EDDHA) and humic substance and of a mixture of humic substance and Fe(o,o‐EDDHA) to determine the dynamics of available Fe. While the mixture did not significantly increase the available soil Fe, it did decrease the rate of Fe retention in the surface soil compared to sole Fe(o,o‐EDDHA). In the field experiment, the substitution in the application solution of 67% of Fe(o,o‐EDDHA) by commercial humic substance increased leaf P in lemon trees from 0.19% with sole Fe(o,o‐EDDHA) to 0.30% and leaf Fe from 94 mg kg^–1 to 115 mg kg^–1. Some quality parameters like vitamin C content and peel thickness were also improved with a partial substitution of Fe(o,o‐EDDHA) by humic substances. We conclude that a partial substitution of commercial Fe chelates by humic substance can improve crop Fe uptake and may thus be economically attractive. The underlying physiological mechanisms and ecological implications require further studies.     

Health-promoting compounds in broccoli as influenced by refrigerated transport and retail sale period

Total aliphatic and indole glucosinolates, phenolic compounds (flavonoids and hydroxycinnamoyl derivatives), and vitamin C contents were evaluated in freshly harvested broccoli (Brassica oleracea L., var. italica, cv. Marathon) inflorescences. These were film-wrapped and stored for 7 days at 1 degrees C to simulate a maximum period of commercial transport and distribution. After cold storage, inflorescences were kept for 3 days at 15 degrees C to simulate a retail sale period. For wrapping, low-density polyethylene (LDPE) of 11 microm thickness was used. Gas composition was about 17% O(2) and 2% CO(2) during cold storage and about 16% O(2) and 3-4% CO(2) during shelf life within packages. The predominant glucosinolates were 4-methylsulfinylbutyl-glucosinolate (glucoraphanin), 3-indolylmethyl-glucosinolate (glucobrassicin), and 1-methoxy-3-indolylmethyl-glucosinolate (neoglucobrassicin). The predominant hydroxycinnamoyl derivatives were identified as 1,2,2'-trisinapoylgentiobiose, 1,2-diferuloylgentiobiose, 1,2'-disinapoyl-2-feruloylgentiobiose, and 3-O-caffeoyl-quinic (neochlorogenic acid). Results showed major losses at the end of both periods, in comparison with broccoli at harvest. Thus, the respective losses, at the end of cold storage and retail periods, were 71-80% of total glucosinolates, 62-59% of total flavonoids, 51-44% of sinapic acid derivatives, and 73-74% caffeoyl-quinic acid derivatives. Slight differences in all compound concentrations between storage and retail sale periods were detected. Distribution and retail periods had minimal effects on vitamin C. Weight loss was monitored at the end of both periods.     

Hydroxytyrosol 4-beta-D-glucoside,an important phenolic compound in olive fruits and derived products

There is increasing interest in olive polyphenols because of their biological properties as well as their contribution to the color, taste, and shelf life of olive products. However, some of these compounds remain unidentified. It has been shown that hydroxytyrosol 4-beta-D-glucoside (4-beta-D-glucosyl-3-hydroxyphenylethanol) coeluted with hydroxytyrosol [(3,4-dihydroxyphenyl)ethanol] under reversed phase conditions in the phenolic chromatograms of olive pulp, vegetation water, and pomace of olive oil processing. A method to separate this compound from hydroxytyrosol by HPLC has been developed. The concentration of this glucoside increased in olive pulp with maturation and could be the main phenolic compound in mature olives. In contrast, the presence of this compound was not detected in olive oil by using HPLC-MS. The compound must be considered both in table olives and olive oil processing because of its glucose and hydroxytyrosol contribution to these products.     

The origin of Manihot esculenta Crantz (Euphorbiaceae)

Summary Contrary to views that cassava (Manihot esculenta) is only known in cultivation an argument is made that wild accessions of the species grow over much of the American neotropics, in Brazil, Bolivia, Peru, Venezuela, Guyana, and Surinam. Three subspecies are recognized. M. esculenta subsp. esculenta is the domesticate and includes all cultivars known in cultivation. The wild M. esculenta subsp. peruviana occurs in eastern Peru and western Brazil. The wild M. esculenta subsp. flabellifolia shows a wider distribution and ranges from the central Brazilian state of Goiás northward to Venezuelan Amazonia. The large area of distribution of the two wild subspecies makes it difficult to assign a place of initial domestication.     

Variation in primitive landraces of common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) from Argentina

The phenotypic variation found in four common bean (Phaseolus vulgaris L.) complex primitive landraces, among a group of accessions collected in Northwestern Argentina in several missions is described, with particular attention to the wide diversity found in some small areas. It is presented a hypothesis about the maintenance of such diversity in bean mixtures or complex primitive landraces that grow close to their wild relative. Wide diversity regarding to seed type and plant characteristics was displayed by the landraces MCM-SV (composed of 11 lines), MCM-292 (14 lines), MCM-298 (5 lines) and VAV-3716 (14 lines). Food uses of dry seed and fresh pod seemed to be more relevant than the aesthetic use although all of them were presumably considered by humans for centuries resulting in the current phenotypes of these complex primitive landraces. Additionally, some weedy types (intermediate between wild and domesticated types) were detected in the landracesMCM-292 and MCM-298. The four complex landraces described consisted of highly diverse mixtures and they could play a role in breeding to enlarge the genetic basis of domesticated bean varieties belonging to the Andean gene pool.     

Polyphenol changes during fermentation of naturally black olives

The individual evolution of phenolic compounds has been studied during the natural fermentation of black olives for the first time. Cyanidin 3-rutinoside and cyanidin 3-glucoside were the main anthocyanins identified in fresh olives, and they were not detected after 1 month of storage either in brine or in olive. The fruit colors were different when aerobic or anaerobic conditions were used and as a consequence of the different anthocyanin polymerizations that took place. At time zero, the polyphenols observed in the olive juice were hydroxytyrosol-4-beta-glucoside, oleuropein, hydroxytyrosol, tyrosol, salidroside, and verbascoside and, after 12 months, the main phenol was hydroxytyrosol. The polyphenol content in the oil phase of olives was also analyzed. The dialdehydic form of elenolic acid linked to hydroxytyrosol and tyrosol, oleuropein aglycon, and ligstroside aglycon were the main compounds found at the beginning of fermentation but were not detected after 3 months. In contrast, hydroxytyrosol, hydroxytyrosol acetate, tyrosol, and tyrosol acetate were the main polyphenols detected in the oil phase of the final product. The acid hydrolysis of the initial glucosides (in olive juice) and the aglycons (in oil phase) was, therefore, the main reaction that took place during fermentation.     

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.     

A comparative study of the precipitation acidity in two cities in Cantabria (Spain) with different degrees of industrialization

A study was carried out on the pH values obtained from bulk (wet and dry) precipitation samples collected simultaneously in two cities in Cantabria (Spain), Santander and Torrelavega over a period of a year. It was observed that the annual weighted average values for the pH of precipitation, 4.42 and 4.21, respectively, differ significantly although the difference is less than that existing between their pollution levels. The difference is greater when total annual H⁺ depositions are compared. The study also demonstrated the strong influence of long-range transport effect on acidity and to a lesser degree the effect of mid-range transport.