Candida Vannini

Proteomic analysis of somatic embryogenesis in Vitis vinifera

Two dimensional gel electrophoresis coupled to mass spectrometry has been used to study the somatic embryogenesis in Vitis vinifera, by comparing embryogenic and non embryogenic calluses of the Thompson seedless cv. More than 1,000 spots were reproducibly resolved in colloidal Coomassie brilliant blue stained gels over a pI nonlinear range of 3–10 in the first dimension and using homogeneous 12.5% polyacrylamide gels in the second dimension. The expression pattern of 35 spots differed significantly between the two samples. These spots were processed by mass spectrometry analysis and the protein identity was assigned by using both the non-redundant protein and EST databases. Several responsive proteins, some already known to be involved in the somatic embryogenesis process while others, for the first time put into relation with this process, have been described. Moreover, they have been subdivided in functional categories, and their putative role is discussed in terms of their relevance in the somatic embryogenesis process.

Morphological and Proteomic Responses of Eruca sativa Exposed to Silver Nanoparticles or Silver Nitrate

Silver nanoparticles (AgNPs) are widely used in commercial products, and there are growing concerns about their impact on the environment. Information about the molecular interaction of AgNPs with plants is lacking. To increase our understanding of the mechanisms involved in plant responses to AgNPs and to differentiate between particle specific and ionic silver effects we determined the morphological and proteomic changes induced in Eruca sativa (commonly called rocket) in response to AgNPs or AgNO3. Seedlings were treated for 5 days with different concentrations of AgNPs or AgNO3. A similar increase in root elongation was observed when seedlings were exposed to 10 mg Ag L1 of either PVP-AgNPs or AgNO3. At this concentration we performed electron microscopy investigations and 2-dimensional electrophoresis (2DE) proteomic profiling. The low level of overlap of differentially expressed proteins indicates that AgNPs and AgNO3 cause different plant responses. Both Ag treatments cause changes in proteins involved in the redox regulation and in the sulfur metabolism. These responses could play an important role to maintain cellular homeostasis. Only the AgNP exposure cause the alteration of some proteins related to the endoplasmic reticulum and vacuole indicating these two organelles as targets of the AgNPs action. These data add further evidences that the effects of AgNPs are not simply due to the release of Ag ions.

Antisense reduction of thylakoidal ascorbate peroxidase in Arabidopsis enhances Paraquat-induced photooxidative stress and Nitric Oxide-induced cell death

The production and characterization of Arabidopsis plants containing a transgene in which the Arabidopsis tAPX is inserted in antisense orientation, is described. tAPX activity in these transgenic tAPX plants is around 50% of control level. The tAPX antisense plants are phenotypically indistinguishable from control plants under normal growth conditions; they show, however, enhanced sensitivity to the O2−-generating herbicide, Paraquat. Interestingly, the tAPX antisense plants show enhanced symptoms of damage when cell death is triggered through treatment with the nitric oxide-donor, SNP. These results are in accordance with the ones recently obtained with transgenic plants overexpressing tAPX; altogether, they suggest that tAPX, besides the known ROS scavenging role, is also involved in the fine changes of H2O2 concentration during signaling events.

AFLP analysis as biomarker of exposure to organic and inorganic genotoxic substances in plants

In recent years several plant species have been in use as bioindicators and several tests have been developed to evaluate the toxicity of environmental pollutants in vegetal organisms. In the present paper Arabidopsis thaliana (L.) Heynh. (ecotype Wassilewskija) was used as bioindicators of two genotoxic substances: potassium dichromate and dihydrophenanthrene. Inhibition of seed germination was observed with both pollutants. AFLP analysis (i) indicated that both substances are genotoxic, (ii) showed that dihydrophenanthrene induces DNA changes in different target sequences than potassium dichromate, (iii) quantified the genotoxic effect using cluster analysis by comparing DNA from treated plants with that of control plants. On the basis of these considerations we suggest that AFLP method is a powerful tool for measuring qualitative and quantitative genotoxic activity due to environmental pollutants. AFLP method can be applied to a wide range of bioindicator organisms and may become a universal methodology to identify target genes for specific genotoxic agents. This could open up possibilities for designing specifically targeted assays and new approaches to risk assessment.

Phytotoxic and genotoxic effects of silver nanoparticles exposure on germinating wheat seedlings

We investigated the effects of 1 and 10 mg L(-1) AgNPs on germinating Triticum aestivum L. seedlings. The exposure to 10 mg L(-1) AgNPs adversely affected the seedling growth and induced morphological modifications in root tip cells. TEM analysis suggests that the observed effects were due primarily to the release of Ag ions from AgNPs. To gain an increased understanding of the molecular response to AgNP exposure, we analyzed the genomic and proteomic changes induced by AgNPs in wheat seedlings. At the DNA level, we applied the AFLP technique and we found that both treatments did not induce any significant DNA polymorphisms. 2DE profiling of roots and shoots treated with 10 mg L(-1) of AgNPs revealed an altered expression of several proteins mainly involved in primary metabolism and cell defense.

Genomic stability in Arabidopsis thaliana transgenic plants obtained by floral dip

The occurrence of DNA modification is an undesired phenomenon accompanying plant cell transformation. The event has been correlated with the stress imposed by the presently utilised transformation procedures, all depending on plant differentiation from in vitro cell culture, but other causes have not been excluded. In this work, transgenic Arabidopsis thaliana plants have been produced by an approach that does not require cell dedifferentiation, being based on in planta Agrobacterium-mediated gene transfer by flower infiltration, which is followed by recovery and selection of transgenic progeny. Genomic DNA changes in transgenic and control plants have been investigated by AFLP and RAMP analysis. Results show no statistically relevant genomic modifications in transgenic plants, as compared with control untreated plants. Variations were observed in callus-derived A. thaliana plants, thus supporting the conclusion that somaclonal variation is essentially correlated with the stress imposed by the in vitro cell culture, rather than with the integration of a foreign gene.

Effects of a complex mixture of therapeutic drugs on unicellular algae Pseudokirchneriella subcapitata

Pharmaceutically-active compounds are regularly and widely released into the aquatic environment in an unaltered form or as metabolites. So far, little is known about their potential detrimental effects on algae populations which can ultimately impact nutrient cycling and oxygen balance. For our analysis, the common microalga Pseudokirchneriella subcapitata (P. subcapitata) was exposed to a mixture of 13 drugs found in Italian wastewaters and rivers. Traces of pharmaceuticals investigated were detected in treated algal cells, except for cyclophosphamide and ranitidine, indicating that these algae are able to absorb pharmaceutical pollutants from the environment. The effects of the treatment were investigated by Amplified Fragment Length Polymorphism (AFLP) assessment of DNA damage and 2-DE proteomic analysis. While no genotoxic effect was detected, proteomic analysis showed that algae are sensitive to the presence of drugs and that, in particular, the chloroplast is affected.

Efficiency of transient transformation in tobacco protoplasts is independent of plasmid amount

We describe an optimized protocol for the transient transformation of tobacco protoplasts mediated by polyethylene–glycol (PEG). As expected, the quantitative β–glucuronidase (Gus) activity driven by pCaMVGus was dependent on the amount of plasmid used. Nevertheless, we demonstrate by an immunodetection method that transformation efficiency did not depend on the amount of plasmid used but on the limitation imposed by cell competence. In fact, we obtained the same percentage of transformed cells (about 60%) using a wide range of plasmid concentrations (0.1–10 μg per test). Finally, we show that, when we used two plasmid types in a mixture at a concentration ranging from 0.1 to 10 μg for each, all transformed cells expressed proteins encoded by both plasmids. Transient expression and co-transformation experiments are routinely used methods and, probably, the major results from this work were assumed by many researchers in this field, but our data experimentally support this assumption.

Uptake and effects of a mixture of widely used therapeutic drugs in Eruca sativa L. and Zea mays L. plants

Pharmaceutically active compounds (PACs) are continuously dispersed into the environment due to human and veterinary use, giving rise to their potential accumulation in edible plants. In this study, Eruca sativa L. and Zea mays L. were selected to determine the potential uptake and accumulation of eight different PACs (Salbutamol, Atenolol, Lincomycin, Cyclophosphamide, Carbamazepine, Bezafibrate, Ofloxacin and Ranitidine) designed for human use. To mimic environmental conditions, the plants were grown in pots and irrigated with water spiked with a mixture of PACs at concentrations found in Italian wastewaters and rivers. Moreover, 10× and 100× concentrations of these pharmaceuticals were also tested. The presence of the pharmaceuticals was tested in the edible parts of the plants, namely leaves for E. sativa and grains for Z. mays. Quantification was performed by liquid chromatography mass spectroscopy (LC/MS/MS). In the grains of 100× treated Z. mays, only atenolol, lincomycin and carbamazepine were above the limit of detection (LOD). At the same concentration in E. sativa plants the uptake of all PACs was >LOD. Lincomycin and oflaxacin were above the limit of quantitation in all conditions tested in E. sativa. The results suggest that uptake of some pharmaceuticals from the soil may indeed be a potential transport route to plants and that these environmental pollutants can reach different edible parts of the selected crops. Measurements of the concentrations of these pharmaceuticals in plant materials were used to model potential adult human exposure to these compounds. The results indicate that under the current experimental conditions, crops exposed to the selected pharmaceutical mixture would not have any negative effects on human health. Moreover, no significant differences in the growth of E. sativa or Z. mays plants irrigated with PAC-spiked vs. non-spiked water were observed.

Exploring the soluble proteome of Tobacco Bright Yellow-2 cells at the switch towards different cell fates in response to heat shocks.

Tobacco (Nicotiana tabacum) Bright Yellow-2 (TBY-2) cells undergo different fates when exposed for 10 minutes to heat stresses of different severity. A 35 degrees C treatment causes a homeostatic response (HRE) allowing cells to cope with the stress; 55 degrees C triggers processes leading to programmed cell death (PCD), which is complete after 72 h. We have used a proteomic approach to gain insight into the molecular mechanisms defining the fate of TBY-2 cells induced by these two heat stresses. Tandem mass spectrometry (MS/MS) and two-dimensional electrophoresis (2-DE) analysis revealed little overlap of differentially-accumulated proteins: the different severities of heat treatment induced the modulation of specific proteins, some of which are responsible for different cell fates. When the imposed heat shock is beyond a certain threshold, the overall reduced metabolism may be the result of a series of events involving gene expression and oxidative damage that would lead to PCD. Our data suggest that the down-accumulation of several proteins involved in cellular redox homeostasis could provide, until now, an unappreciated contribution to understanding how many partners are involved in promoting the redox impairment leading to PCD. Moreover post-translational modifications seem to play important regulatory roles in the adaptation of TBY-2 cells to different intensities of heat stress.