Eliana Nutricati

Clonothrix fusca Roze 1896, a Filamentous, Sheathed, Methanotrophic γ-Proteobacterium

ABSTRACT Crenothrix polyspora Cohn 1870 and Clonothrix fusca Roze 1896 are two filamentous, sheathed microorganisms exhibiting complex morphological differentiation, whose phylogeny and physiology have been obscure for a long time due to the inability to cultivate them. Very recently, DNA sequencing data from uncultured C. polyspora-enriched material have suggested that Crenothrix is a methane-oxidizing γ-proteobacterium (39). In contrast, the possible ecological function of C. fusca, originally considered a developmental stage of C. polyspora, is unknown. In this study, temporal succession of two filamentous, sheathed microorganisms resembling Cohns Crenothrix and Rozes Clonothrix was observed by analyzing the microbial community of an artesian well by optical microscopy. Combined culture-based and culture-independent approaches enabled us to assign C. fusca to a novel subgroup of methane-oxidizing γ-proteobacteria distinct from that of C. polyspora. This assignment was supported by (i) methane uptake and assimilation experiments, (ii) ultrastructural data showing the presence in C. fusca cytoplasm of an elaborate membrane system resembling that of methanotrophic γ-proteobacteria, and (iii) sequencing data demonstrating the presence in its genome of a methanol dehydrogenase α subunit-encoding gene (mxaF) and a conventional particulate methane mono-oxygenase α subunit-encoding gene (pmoA) that is different from the unusual pmoA (u-pmoA) of C. polyspora.

Characterization of two Arabidopsis thaliana glutathione S-transferases

Glutathione S-transferases (GST) are multifunctional proteins encoded by a large gene family, divided on the basis of sequence identity into phi, tau, theta, zeta and lambda classes. The phi and tau classes are present only in plants. GSTs appear to be ubiquitous in plants and are involved in herbicide detoxification and stress response, but little is known about the precise role of GSTs in normal plant physiology and during biotic and abiotic stress response. Two cDNAs representing the two plant classes tau and phi, AtGSTF9 and AtGSTU26, were expressed in vitro and the corresponding proteins were analysed. Both GSTs were able to catalyse a glutathione conjugation to 1-chloro-2,4-dinitrobenzene (CDNB), but they were inactive as transferases towards p-nitrobenzylchloride (pNBC). AtGSTF9 showed activity towards benzyl isothiocyanate (BITC) and an activity as glutathione peroxidase with cumene hydroperoxide (CumHPO). AtGSTU26 was not active as glutathione peroxidase and towards BITC. RT-PCR analysis was used to evaluate the expression of the two genes in response to treatment with herbicides and safeners, chemicals, low and high temperature. Our results reveal that AtGSTU26 is induced by the chloroacetanilide herbicides alachlor and metolachlor and the safener benoxacor, and after exposure to low temperatures. In contrast, AtGSTF9 seems not to be influenced by the treatments employed.

Biochemical characterization of a lipase from olive fruit (Olea europaea L.)

Lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) is the first enzyme of the degradation path of stored triacylglycerols (TAGs). In olive fruits, lipase may determine the increase of free fatty acids (FFAs) which level is an important index of virgin olive oil quality. However, despite the importance of virgin olive oil for nutrition and human health, few studies have been realized on lipase activity in Olea europaea fruits. In order to characterize olive lipase, fruits of the cv. Ogliarola, widely diffused in Salento area (Puglia, Italy), were harvested at four stages of ripening according to their skin colour (green, spotted I, spotted II, purple). Lipase activity was detected in the fatty layer obtained after centrifugation of the olive mesocarp homogenate. The enzyme exhibited a maximum activity at pH 5.0. The addition of calcium in the lipase assay medium leads to an increment of activity, whereas in the presence of copper the activity was reduced by 75%. Furthermore, mesocarp lipase activity increases during olive development but declined at maturity (purple stage). The data represent the first contribution to the biochemical characterization of an olive fruit lipase associated to oil bodies.

Influence of Environmental Factors on Essential Oil Variability in Thymbra capitata (L.) Cav. Growing Wild in Southern Puglia (Italy)

Abstract The composition of officinal plants in terms of active principles exhibits high intraspecific variability which is determined not only genetically but also by environmental conditions, growth area and harvesting time. This study analyzed the essential oil variability in Thymbra capitata (L.) Cav. in specimens growing wild in 23 places in Southern Puglia (Italy). The results show high chemical polymorphism in the oil content of T. capitata. Application of the spatial autocorrelation index to the principal components highlighted a relationship between thermal parameters and the composition in terms of active principles. The carvacrol chemotype was only present under the hottest and driest conditions. These results show that not only is the biosynthetic pathway of phenolic monoterpenes in T. capitata favored in high-temperature environments, as reported in other Lamiaceae, but also that carvacrol is present only in markedly “Mediterranean-like” environments.

Arthrinium phaeospermum isolated from Tuber borchii ascomata: the first evidence for a “Mycorrhization Helper Fungus”?

A filamentous fungus was isolated from Tuber borchii Vitt. fruiting bodies, and it was identified as an Arthrinium phaeospermum (Corda) M.B. Ellis strain, an “endophyte” that forms various associations with healthy leaves, stems, and roots of plants. Molecular analysis confirmed the association of this filamentous fungus with the ascocarps of all collection sites in Salento, Apulia (South Italy). An in vitro symbiosis system between Cistus creticus L. and T. borchii was set up; A. phaeospermum appears to be able to promote mycorrhiza formation in Cistus seedlings, inducing primary root shortening and an increase of secondary roots, similar to the effect of Mycorrhization Helper Bacteria (MHB). Compartmented and uncompartmented bioassays were carried out to investigate the effects of exudates/volatiles released by the truffle-hosted fungus on root architecture; the results showed root shortening in compartmented bioassay suggesting that volatiles released by the fungus alone are sufficient to alter root morphology in early phase of interaction before the mycorrhiza formation. The first evidence for an influence of a truffle-hosted fungus on ectomycorrhizal symbiosis establishment is reported.

Phylogenetic analysis of viruses in Tuscan Vitis vinifera sylvestris (Gmeli) Hegi

The health status of the native grapevine Vitis vinifera subsp. sylvestris (Gmeli) Hegi in natural areas in Europe has received little attention. A survey was carried out on wild grapevines in Tuscany (Italy), where isolates of the Grapevine rupestris stem pitting virus (GRSPaV), Grapevine leafroll-associated virus 1 and 3 (GLRaV-1 and GLRaV-3) and Grapevine virus A (GVA) were detected. The complete coat protein (CP) region of these isolates was sequenced to investigate the relationship of the viral variants from Tuscan wild grapevines with isolates from different geographical origins. According to the phylogenetic analyses, GLRaV-1 and GLRaV-3 isolates from Tuscan wild grapevines clustered with isolates from cultivated grapevines with nucleotide sequence identities ranging from 66% to 87% and from 72.5% to 99% respectively, without any correlation between the distribution and geographical origin. Conversely, GRSPaV and GVA isolates clustered together with other Italian isolates from V. vinifera with nucleotide sequence identities ranging from 71.14% to 96.12% and from 73.5% to 92%, respectively. Our analysis of the whole amino acid sequences revealed a high conservation level for the studied proteins explained by a selective pressure on this genomic region, probably due to functional constraints imposed on CP, such as specific interactions with cellular receptors in the insect vectors necessary for successful transmission. In addition, analyses of genetic recombination suggest no significant point mutations that might play a significant role in genetic diversification. The dN/dS ratio also estimated a low number of non-silent mutations, highlighting the purifying selective pressure. The widespread distribution of the Rugose wood complex (GRSPaV and GVA associated disease) in comparison with the Grapevine Leafroll associated viruses (GLRaV-1 and -3) could explain the major geographical correlation found for the viral variants detected in Tuscany.

Salvia clandestina L.: unexploited source of danshensu

Abstract We report the characterisation of Salvia clandestina L. shoots and roots aqueous extract using HPLC-ESI/MS-TOF. Among the 29 compounds detected, we observed the presence of danshensu (3-(3,4-dihydroxy-phenyl) 2-hydroxy-propinic acid), a powerful antioxidant and a cardio-protective agent. The danshensu content found in the shoots of S. clandestina was considerable (4.96 mg g−1 DW) if compared with previous studies on S. miltiorrhiza Bunge, the main source of this compound. We also determined the total phenolic concentration and we evaluated their antioxidant activity by ABTS, FRAP and Superoxide anion scavenging methods. All the three assays confirmed a greater antioxidant activity for the shoots in comparison to roots. S. clandestina shoots may represent a valuable and natural unexploited source of danshensu and other phenolic compounds, so that it may be useful for future applications in functional foods and pharmaceutical industries.

Glutathione S-transferase related detoxification processes are correlated with receptor-mediated vacuolar sorting mechanisms

Key messageTriticum durumGlutathioneS-transferase Z1 is specifically responsive to glyphosate. Its expression influences the receptor-mediated vacuolar sorting mechanisms involved in tolerance mechanisms.AbstractA zeta subfamily glutathione S-transferase gene from Triticum durum (cv Cappelli) (TdGSTZ1) was characterized as part of a complex detoxification mechanism. The effect of different abiotic stresses on TdGSTZ1 revealed that the gene is unexpectedly responsive to glyphosate (GLY) herbicide despite it should not be part of tolerance mechanisms. Its role in the non-target-site mechanism of GLY resistance was then investigated. To analyze the GLY and the TdGSTZ1 overexpression effects on vacuolar sorting mechanisms, we performed transient transformation experiments in Nicotiana tabacum protoplasts using two vacuolar markers, AleuGFPgl133 and GFPgl133Chi, labeling the Sar1 dependent or independent sorting, respectively. We observed that the adaptive reaction of tobacco protoplasts vacuolar system to the treatment with GLY could be partially mimicked by the overexpression of TdGSTZ1 gene. To confirm the influence of GLY on the two vacuolar markers accumulation and the potential involvement of the secretion pathway activity in detoxification events, Arabidopsis thaliana transgenic plants overexpressing the non-glycosylated versions of the two markers were analyzed. The results suggested that GLY treatment specifically altered different vacuolar sorting characteristics, suggesting an involvement of the receptor-mediated AleuGFP sorting mechanism in GLY resistance. Finally, the expression analysis of selected genes confirmed that the non-target-site GLY resistance mechanisms are related to vacuolar sorting.

Iris revoluta Colas., natural hybrid origin species: characterization and preservation problems

Abstract In order to preserve ex situ rare or vulnerable species, it is necessary to characterise their reproductive mechanisms, and to locate the origin of the interference with their development in their natural habitat. Iris revoluta Colas. is a very rare endemic species of the Salento peninsula (Italy), showing scarce sexual reproduction, but good vegetative propagation through rhizomes, as many other Iris species of natural hybrid origin. For many years this Iris has been cultivated in the Botanical Garden of the Di.S.Te.B.A., University of Lecce, where a detailed study has been initiated to investigate the phenomena that may limit its sexual reproduction.