Impact of TiO2 nanoparticles on Vicia narbonensis L.: potential toxicity effects.

This work was aimed to provide further information about toxicology of TiO2 nanoparticles (NPs) on Vicia narbonensis L., considering different endpoints. After exposure to TiO2 nanoparticle suspension (mixture of rutile and anatase, size <100 nm) at four different concentrations (0.2, 1.0, 2.0 and 4.0 ‰), the seeds of V. narbonensis were let to germinate in controlled environmental conditions. After 72 h, the extent of the success of the whole process (seed germination plus root elongation) was recorded as the vigour index, an indicator of possible phytotoxicity. After the characterisation of the hydric state of different materials, oxidative stress and enzymatic and nonenzymatic antioxidant responses were considered as indicators of possible cytotoxicity and to assess if damage induced by TiO2 NPs was oxidative stress-dependent. Cytohistochemical detection of in situ DNA fragmentation as genotoxicity endpoint was monitored by TUNEL reaction. The treatments with TiO2 NPs in our system induced phytotoxic effects, ROS production and DNA fragmentation. The nonenzymatic and enzymatic antioxidant responses were gradually and differentially activated and were able to maintain the oxidative damage to levels not significantly different from the control. On the other hand, the results of DNA fragmentation suggested that the mechanisms of DNA repair were not effective enough to eliminate early genotoxicity effects.

Measuring PLD activity in vivo.

Phospholipase D (PLD) hydrolyzes structural phospholipids like phosphatidylcholine (PC) and phosphatidylethanolamine (PE) into phosphatidic acid (PA) and free choline/ethanolamine. In plants, this activity can be stimulated by a wide variety of biotic and abiotic stresses (Li et al., Biochim Biophys Acta 1791:927-935, 2009; Testerink and Munnik, J Exp Bot 62(7):2349-2361, 2011). This chapter describes a protocol for the measurement of PLD activity in vivo. The protocol takes advantage of a unique property of PLD, i.e., its ability to substitute a primary alcohol, such as 1-butanol, for water in the hydrolytic reaction. This transphosphatidylation reaction results in the formation of phosphatidylbutanol (PBut), which is a specific and unique reporter for PLD activity. The assay is highly sensitive for detecting PLD activity in vivo, following stimulation of intact plant cells, seedlings, and tissues, being a valuable method for studying the regulation of plant PLD activity in vivo.

Expression, detection of candidate function and homology modeling for Vicia villosa ornithine δ-aminotransferase.

The accumulation of compatible solutes during stress in plant cell is well documented. Proline is one of these solutes which accumulate in the cytosol in response to drought or salinity stress in plants. Proline has several functions during stress just like osmotic adjustment, osmoprotection, free radical scavenger and antioxidant. Ornithine δ-aminotransferase (δ-OAT) is an important enzyme in proline biosynthetic pathway. It catalyzes the transamination of ornithine to pyrroline-5-carboxylate which can be reduced into proline. Expression of ornithine δ-aminotransferase gene isolated from Vicia villosa (VvOAT) showed protein with a molecular mass of 63 KDa which is compatible with the predicted mass and after VvOAT gene delivery into E. coli host HB101, VvOAT gene enhanced its salt tolerance. Homology modeling of VvOAT was performed based on the crystal structure of the ornithine δ-aminotransferase from humans (PDB code 2OATA). With this model, a flexible docking study with the substrate and inhibitors was performed. The results indicated that PHE170 and ASN171 in VvOAT are the important determinant residues in binding as they have strong hydrogen bonding contacts with the substrate and inhibitors. All the obtained results indicated the efficiency of utilizing this gene in conferring salt tolerance.

Vicianin hydrolase is a novel cyanogenic beta-glycosidase specific to beta-vicianoside (6-O-alpha-L-arabinopyranosyl-beta-D-glucopyranoside) in seeds of Vicia angustifolia.

The cyanogenic disaccharide glycoside, vicianin [mandelonitrile beta-vicianoside (6-O-alpha-L-arabinopyranosyl-beta-D-glucopyranoside)], is accumulated in seeds of Vicia angustifolia var. segetalis. Vicianin hydrolase (VH) catalyzes the hydrolysis of vicianin into mandelonitrile and a disaccharide vicianose. VH was purified from the seeds using DEAE-, CM- and Con A-Sepharose chromatography, and the molecular mass of the purified VH was estimated to be 56 kDa on SDS-PAGE. The N-terminal amino acid sequence of the purified VH was determined, and a cDNA encoding VH was obtained. The deduced VH protein consists of a 509 amino acid polypeptide containing a putative secretion signal peptide. It shares about 50% identity with various kinds of plant beta-glycosidases including tea leaf beta-primeverosidase and furcatin hydrolase, and is classified in family 1 of the glycosyl hydrolases. The VH transcript was detected abundantly in seeds and moderately in flowers, but only slightly in leaves, stems and roots, indicating that the organ distribution of VH expression is similar to that of the substrate vicianin. The recombinant VH was produced in insect cells with a baculovirus system, and was compared with the native VH in terms of substrate specificity. Both enzymes hydrolyzed vicianin to release vicianose, demonstrating that VH is a disaccharide-specific beta-glycosidase. VH also hydrolyzed the mandelonitrile beta-glucoside prunasin to some extent but did not hydrolyze the gentiobioside amygdalin, both of which contain the same aglycone as vicianin. Thus, VH is a unique cyanogenic glycosidase showing high glycone specificity for the disaccharide vicianoside.

Isozyme variation and phylogenetic relationships in Vicia subgenus Cracca (Fabaceae).

BACKGROUND AND AIMS: The phylogenetic relationships among 27 vetch species belonging to the subgenus Cracca of the genus Vicia were studied in comparison with three species of Lathyrus section Lathyrus on the basis of isozyme variation. METHODS: Isozymes encoded by 15 putative loci of ten enzymes were resolved by polyacrylamide gel electrophoresis and isozyme variation was analysed by using parsimony and neighbour-joining methods. KEY RESULTS: The analyses revealed 63 parsimony-informative and 36 species-specific orthozymes. Of the latter, 23 are monomophic and are suitable for identification of V. benghalensis, V. palaestina, V. dumetorum, V. pisiformis, V. sylvatica, V. onobrychioides, V. cappadocica, V. cretica, V. articulata, V. tetrasperma, V. ervilia, V. hirsuta and V. loiseleurii. Polymorphism with heterozygous and homozygous isozyme genotypes was found for V. cracca, V. tenuifolia, V. ochroleuca, V. villosa, V. sylvatica, V. cassubica, V. sparsiflora, V. megalotropis, V. altissima, V. onobrychioides, V. cassia, V. cretica and L. heterophyllus, reflecting outcrossing in these species. By contrast, V. benghalensis, V. palaestina, V. disperma, V. dumetorum, V. pisiformis, V. orobus, V. pauciflora, V. tetrasperma and V. loiseleurii had only homozygous isozyme genotypes at polymorphic loci. Isozyme-based phylogenetic trees are presented. CONCLUSIONS: Sections Cracca, Ervum, Pedunculatae and Lenticula of traditional taxonomy are monophyletic groups, whereas sections Oroboideae (= Vicilla) and Panduratae appear polyphyletic and section Cassubicae is split into two species-couples linked at a low level of support. Treatment of ervoid species in a separate subgenus Ervum is not supported because of its polyphyly.