Effect of Interfacial SiOx Defects on the Functional Properties of Si-Transition Metal Oxide Photoanodes for Water Splitting.

The transfer of photogenerated charges through interfaces in heterojunction photoanodes is a key process that controls the efficiency of solar water splitting. Considering Co3O4/SiOx/Si photoanodes prepared by physical vapor deposition as a representative case study, it is shown that defects normally present in the native SiOx layer dramatically affect the onset of the photocurrent. Electron paramagnetic resonance indicates that the signal of defects located in dangling bonds of trivalent Si atoms at the Si/SiOx interface vanishes upon vacuum annealing at 850 °C. Correspondingly, the photovoltage of the photoanode increases to ≈500 mV. Similar results are obtained for NiO/SiOx/Si photoanodes. Photoelectrochemical analysis and impedance spectroscopy (in solution and in the solid state) indicate how the defect annealing modifies the Co3O4/SiOx/Si junction. This work shows that defect annealing at the solid-solid interface in composite photoanodes strongly improves the efficiency of charge transfer through interfaces, which is the basis for effective solar-to-chemical energy conversion.

Seed priming: state of the art and new perspectives.

Priming applied to commercial seed lots is widely used by seed technologists to enhance seed vigour in terms of germination potential and increased stress tolerance. Priming can be also valuable to seed bank operators who need improved protocols of ex situ conservation of germplasm collections (crop and native species). Depending on plant species, seed morphology and physiology, different priming treatments can be applied, all of them triggering the so-called 'pre-germinative metabolism'. This physiological process takes place during early seed imbibition and includes the seed repair response (activation of DNA repair pathways and antioxidant mechanisms), essential to preserve genome integrity, ensuring proper germination and seedling development. The review provides an overview of priming technology, describing the range of physical-chemical and biological treatments currently available. Optimised priming protocols can be designed using the 'hydrotime concept' analysis which provides the theoretical bases for assessing the relationship between water potential and germination rate. Despite the efforts so far reported to further improve seed priming, novel ideas and cutting-edge investigations need to be brought into this technological sector of agri-seed industry. Multidisciplinary translational research combining digital, bioinformatic and molecular tools will significantly contribute to expand the range of priming applications to other relevant commercial sectors, e.g. the native seed market.

DNA profiling, telomere analysis and antioxidant properties as tools for monitoring ex situ seed longevity.

BACKGROUND AND AIMS: The germination test currently represents the most used method to assess seed viability in germplasm banks, despite the difficulties caused by the occurrence of seed dormancy. Furthermore, seed longevity can vary considerably across species and populations from different environments, and studies related to the eco-physiological processes underlying such variations are still limited in their depth. The aim of the present work was the identification of reliable molecular markers that might help in monitoring seed deterioration. METHODS: Dry seeds were subjected to artificial ageing and collected at different time points for molecular/biochemical analyses. DNA damage was measured using the RAPD (random amplified polymorphic DNA) approach while the seed antioxidant profile was obtained using both the DPPH (1,1-diphenyl, 2-picrylhydrazyl) assay and the Folin-Ciocalteu reagent method. Electron paramagnetic resonance (EPR) provided profiles of free radicals. Quantitative real-time polymerase chain reaction (QRT-PCR) was used to assess the expression profiles of the antioxidant genes MT2 (type 2 metallothionein) and SOD (superoxide dismutase). A modified QRT-PCR protocol was used to determine telomere length. KEY RESULTS: The RAPD profiles highlighted different capacities of the two Silene species to overcome DNA damage induced by artificial ageing. The antioxidant profiles of dry and rehydrated seeds revealed that the high-altitude taxon Silene acaulis was characterized by a lower antioxidant specific activity. Significant upregulation of the MT2 and SOD genes was observed only in the rehydrated seeds of the low-altitude species. Rehydration resulted in telomere lengthening in both Silene species. CONCLUSIONS: Different seed viability markers have been selected for plant species showing inherent variation of seed longevity. RAPD analysis, quantification of redox activity of non-enzymatic antioxidant compounds and gene expression profiling provide deeper insights to study seed viability during storage. Telomere lengthening is a promising tool to discriminate between short- and long-lived species.

Spore-forming bacteria in soil cultivated with GM white poplars: isolation and characterization.

The impact of transgenic white poplars (Populus alba L. cv. 'Villafranca') was assessed on the soil aerobic spore-forming bacteria (SFB). The genetically modified poplars, expressing either the StSy gene for resveratrol production or the bar gene for herbicide tolerance, were cultivated in greenhouse. The occurrence of SFB was monitored in soil samples collected at eight different timepoints over a two-year period. The total culturable bacterial population of the StSy and bar trials underwent significant seasonal fluctuations in the range of 10(6)-2.5 x 10(8) CFU/g dry soil and of 10(4)-5 x 10(8) CFU/g dry soil, respectively. Changes occurred also within the culturable SFB population with size varying at 10(3)-5 x 10(4) CFU/g dry soil and 10(2)-2 x 10(5) CFU/g dry soil in the StSy and bar trials, respectively. No significant differences in the size of the total and SFB culturable populations were observed when comparing each transgenic line with the nontransformed control line while seasonal shifts of soil bacterial populations were evident in both trials. The culturable SFB fraction included three isolates (SFB-1, SFB-2 and SFB-3) classified by 16S rDNA sequence analysis as members of the Bacillus genus. According to the reported data, cultivation of both herbicide-resistant and resveratrol-producing GM white poplars did not affect the culturable SFB population at the soil level.

Response to UV-C radiation in topo I-deficient carrot cells with low ascorbate levels.

In animal cells, recent studies have emphasized the role played by DNA topoisomerase I (topo I) both as a cofactor of DNA repair complexes and/or as a damage sensor. All these functions are still unexplored in plant cells, where information concerning the relationships between DNA damage, PCD induction, and topo I are also limited. The main goal of this study was to investigate the possible responses activated in topo I-depleted plant cells under oxidative stress conditions which induce DNA damage. The carrot (Daucus carota L.) AT1-beta/22 cell line analysed in this study (characterized by an antisense-mediated reduction of top1beta gene expression of approximately 46% in association with a low ascorbate content) was more sensitive to UV-C radiation than the control line, showing consistent cell death and high levels of 8-oxo-dG accumulation. The topo I-depleted cells were also highly susceptible to the cross-linking agent mitomycin C. The death response was associated with a lack of oxidative burst and there were no changes in ascorbate metabolism in response to UV-C treatment. Electron and fluorescence microscopy suggested the presence of three forms of cell death in the UV-C-treated AT1-beta/22 population: necrosis, apoptotic-like PCD, and autophagy. Taken together, the data reported here support a reduced DNA repair capability in carrot topo I-deficient cells while the putative relationship between topo I-depletion and ascorbate impairment is also discussed.

Production of transgenic barrel medic (Medicago truncatula Gaernt.) using the ipt-type MAT vector system and impairment of Recombinase-mediated excision events.

Expression of the uidA reporter gene was tested in transformation experiments of barrel medic (Medicago truncatula Gaertn.) with the ipt-type control vectors pIPT5, pIPT10 and pIPT20 and distinct in vitro culture conditions. The highest GUS expression levels were obtained with the pIPT10 construct carrying the ipt gene under the control of the native ipt promoter and using kanamycin as selective agent. The ipt-shooty transformants, characterized by the absence of both rooting ability and apical dominance associated with vitrification, were easily identified by visual selection. Using only the ipt gene as selectable marker, we obtained a stable transformation frequency of 9.8% with pIPT10 construct. The ipt-type MAT vector pEXM2 was then used to monitor the excision events mediated by the yeast Recombinase and the consequent production of ipt marker-free transgenic plants. Transgenic ipt-shooty lines were recovered at a frequency of 7.9% in the absence of kanamycin-based selection. The ipt-shooty phenotype was maintained in all the transgenic lines and no reversion to the normal phenotype occurred. PCR analysis revealed the presence of the 'hit and run' cassette in the genome of all the regenerated ipt-shooty lines while RT-PCR experiments confirmed the expression of the R gene, encoding the yeast Recombinase. A detailed molecular investigation, carried out to verify the integrity of the RS sites, revealed that these regions were intact in most cases. Our results with barrel medic suggest that the MAT system must be carefully evaluated and discussed on a case by case basis.

Methyl jasmonate disrupts shoot formation in tobacco thin cell layers by over-inducing mitotic activity and cell expansion.

The aim of the present study was to determine early cyto-histological events associated with the reduced number of shoots formed at the end of culture in tobacco (Nicotiana tabacum L.) thin cell layers treated with methyl jasmonate (MJ) [S. Biondi et al. (2001) J Exp Bot 52:1-12]. The results show that 0.1-10 microM MJ strongly stimulated mitotic activity early in culture relative to untreated controls. Treatment with MJ also induced anomalous mitoses. Enhanced proliferative growth was confirmed by northern analysis and in situ hybridisation using cDNA probes of the G1/S phase-specific genes ubiquitin carboxyl-extension protein (ubi-CEP), topoisomerase 1 (top1) and ribonucleotide reductase (RNR). The formation of meristematic cell clusters on day 5 was also enhanced by 1 muM MJ, but subsequent development of these cell clusters into meristemoids and shoot primordia was reduced by all MJ concentrations in a dose-dependent manner. Cell expansion was stimulated by MJ concentrations ranging from 0.001 to 10 microM; expanded cells frequently occurred around and within meristemoids and shoot primordia, and displayed thickened and suberised cell walls; cell wall thickness increased with increasing MJ concentration. These cytological events caused alterations in the tunica and stem differentiation of the shoot dome. The apparently paradoxical role of MJ, which deregulates shoot formation through a stimulation of growth events, i.e., mitotic activity and cell expansion, is discussed.

Isolation and characterization of photosystem II subcomplexes from cyanobacteria lacking photosystem I.

A photosystem II (PSII) core complex lacking the internal antenna CP43 protein was isolated from the photosystem II of Synechocystis PCC6803, which lacks photosystem I (PSI). CP47-RC and reaction centre (RCII) complexes were also obtained in a single procedure by direct solubilization of whole thylakoid membranes. The CP47-RC subcore complex was characterized by SDS/PAGE, immunoblotting, MALDI MS, visible and fluorescence spectroscopy, and absorption detected magnetic resonance. The purity and functionality of RCII was also assayed. These preparations may be useful for mutational analysis of PSII RC and CP47-RC in studying primary reactions of oxygenic photosynthesis.

Spatial expression of DNA topoisomerase I genes during cell proliferation in Daucus carota.

The spatial expression of carrot (Daucus carota L.) top1 genes encoding the two isoforms of the enzyme DNA topoisomerase I (EC 5.99.1.2) was investigated. In situ hybridization analysis performed with a probe recognizing both top1 transcripts provided evidence that in explanted hypocotyls induced to proliferate in vitro by the addition of the growth regulator 2,4-dichlorophenoxyacetic acid (2,4-D), the mRNA accumulation parallels the proliferation of provascular cells of the stelar cylinder. During somatic embryogenesis, the histological distribution of top1 transcripts was strongly evident at the stage of torpedo-shaped embryos, but gene expression was not only restricted to meristematic regions. When the spatial localization was extended to carrot vegetative apices and the investigation was carried out with specific probes for top1alpha and top1beta, both transcripts preferentially accumulated in tissues having mitotic activity.

Influence of the axial ligands on the spectral properties of P700 of photosystem I: a study of site-directed mutants.

Two histidines provide the axial ligands of the two chlorophyll a (Chl a) molecules which form the primary electron donor (P700) of photosystem I (PSI). Histidine 676 in the protein subunit PsaA, His(A676), and histidine 656 in subunit PsaB, His(B656), were replaced in the green algae Chlamydomnas reinhardtii by site-directed mutagenesis with nonpolar, uncharged polar, acidic, and basic amino acid residues. Only the substitutions with uncharged polar residues led to a significant accumulation of PSI in the thylakoid membranes. These PSI complexes were isolated and the physical properties of the primary donor characterized. The midpoint potential of P700(+)(*)/P700 was increased in all mutants (up to 140 mV) and showed a dependence on size and polarizability of the residues when His(B656) was substituted. In the light-minus-dark absorbance spectra, all mutations in PsaB exhibited an additional bleaching band at 665 nm at room temperature comparable with the published spectrum for the replacement of His(B656) with asparagine [Webber, A. N., Su Hui, Bingham, S. E., Käss, H., Krabben, L., Kuhn, M., Jordan, R., Schlodder, E., and Lubitz, W. (1996) Biochemistry 35, 12857-12863]. Substitutions of His(A676) showed an additional shoulder around 680 nm. In the low-temperature absorbance difference spectra of P700(+)(*)/P700, a blue shift of the main bleaching band by 2 nm and some changes in the spectral features around 660 nm were observed for mutations of His(B656) in PsaB. The analogous substitution in PsaA showed only a shift of the main bleaching band. Similar effects of the mutations were found in the (3)P700/P700 absorbance difference spectra at low temperatures (T = 2 K). The zero-field splitting parameters of (3)P700 were not significantly changed in the mutated PSI complexes. The electron spin density distribution of P700(+)(*), determined by ENDOR spectroscopy, was only changed when His(B656) was replaced. In all measurements, two general observations were made. (i) The replacement of His(B656) had a much stronger impact on the physical properties of P700 than the mutation of His(A676). (ii) The exchange of His(B656) with glutamine induces the smallest changes in the spectra or the midpoint potential, whereas the other replacements exhibited a stronger but very similar influence on the spectroscopic features of P700. The data provide convincing evidence that the unpaired electron in the cation radical and the triplet state of P700 are mainly localized on the Chl a of the dimer which is axially coordinated by His(B656).

Carrot cells contain two top1 genes having the coding capacity for two distinct DNA topoisomerases I.

Five DNA topoisomerase I cDNA clones were isolated from a carrot (Daucus carota) cDNA library and two classes of nucleotide sequences were found. One component of the first class, pTop9, perfectly matches the open reading frame of pTop28, a truncated top1 cDNA previously described, and extended it by 594 nucleotides (top1alpha). A member of the second class, pTop11, contains an open reading frame 2727 bp long (top1ss) with a coding capacity for a second putative DNA topoisomerase I of 101 kDa. Both pTop9 and pTop11 clones are full length cDNAs. The two deduced amino acid sequences share a relevant similarity (89%) only at the C-terminal domain, whereas the similarity is reduced to 32% in the N-terminal region. Southern blot analysis and PCR amplification of genomic DNAs from carrot pure lines suggested the presence of two distinct loci. Northern blot analysis revealed the presence of two distinct transcripts of 3.0 and 3.2 kb in both cycling and starved cell populations. Three fusion peptides corresponding to the N-terminal domain of the alpha and ss forms and from the common C-terminal domain of carrot topoisomerases I were overexpressed in E. coli cells and used to raise antibodies in rabbit. Immunolocalization seems to suggest the presence of two topoisomerases I in carrot nuclei.

Cloning of a cDNA encoding DNA topoisomerase I in Daucus carota and expression analysis in relation to cell proliferation.

DNA topoisomerase I is an enzyme involved in several processes related to DNA metabolism. Despite the physiological importance, the regulation of top1 gene expression has not yet been investigated in plants. In order to monitor the possible correlation between levels of top1 transcripts and the proliferative state of the cell, two partially overlapping cDNAs encoding DNA topoisomerase I from Daucus carota have been isolated from a poly(A)(+)-primed library, using an Arabidopsis thaliana probe, and from a cDNA library spanning the 5' region of the top1 transcript, which was constructed using an antisense specific oligonucleotide. The top1 nucleotide sequence encoded an open reading frame of 2370 bp, predicting a protein of 90 kDa. The deduced amino acid sequence showed a similarity of 51% with A. thaliana, 41% with S. cerevisiae, 40% with S. pombe and 31% with H. sapiens, respectively. Southern blot analysis, performed under moderate stringency conditions, showed the presence of a single-copy gene. Evaluation of the top1 mRNA steady-state level revealed, besides a constitutive expression in vegetative carrot tissues, an induced expression related to cell proliferation.

A well resolved ODMR triplet minus singlet spectrum of P680 from PSII particles.

An ADMR T-S spectrum of the primary donor (P680) of photosystem II (PSII) was obtained from anaerobically photoreduced particles. The spectrum is the best resolved obtained so far having a main bleaching band at 684 nm with a linewidth of only 100 cm-1. The view that this spectrum is produced by native homogeneous P680 unlike those obtained before is defended. A small bleaching observed at 678 nm is discussed in terms of the reaction center structure. One possible interpretation of the observations is that P680 is a very loose dimer with an exciton splitting of only 144 cm-1 corresponding to a dimer center-to-center distance of roughly 11.5 A.

Proteolytic and partial sequencing studies of the bifunctional dihydrofolate reductase-thymidylate synthase from Daucus carota.

The bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) of Daucus carota has been further characterized as regards molecular weight, amino acid composition, protease digestion and microsequencing of proteolytic peptides. Data reported in this paper demonstrate that the carrot protein has a calculated Mr of 124,000 thus indicating that, contrarily to what has previously been suggested, it occurs as a dimer of identical subunits. Results of partial amino acid microsequencing show the presence of sequences highly homologous with those of the active sites of both DHFR and TS from other organisms confirming, at the structural level, the bifunctional nature of the carrot protein. As in the case of Leishmania tropica DHFR-TS, incubation of the carrot protein with V8 protease led to a rapid loss of TS activity while retaining that of DHFR. However the pattern of proteolysis did not allow to establish whether the sequence of domains is DHFR-TS as in Leishmania, or vice versa. Low homology of other amino acid sequences, as judged by computer analysis, and absence of common epitopes indicate an apparent divergence between carrot and leishmanian proteins.

Cellular localization of dihydrofolate reductase-thymidylate synthase in carrot cells.

The intracellular distribution and localization of dihydrofolate reductase-thymidylate synthase of wild type suspension carrot cells was analysed using cytochemical and immunocytochemical techniques; in both resting and growing normal cells (E4) the activity appeared to be predominantly cytoplasmic. The pattern of localization of the enzyme was also analysed during the different phases of the cell cycle. To this end carrot cells were synchronized with aphidicolin (an inhibitor of the alpha-like DNA polymerase which blocks cells at the G1/S boundary) and cycle phases checked by labelled-thymidine incorporation. Protoplasts obtained from cells inhibited with aphidicolin or from cells sampled at different times after the removal of the drug (S and G2 phase), failed to show a nuclear localization of DHFR-TS. These results indicate that in carrot the bifunctional enzyme does not change compartment during the cell cycle. Surprisingly Mtx-resistant cells (E2A2, E2A1C6; overproducing DHFR-TS) showed, irrespective of their physiological state (quiescent or growing), also a relevant nuclear or perinuclear immunofluorescence which could not be detected using cytochemical techniques. The reason of this altered localization is not clear and its possible relation with altered cytophysiological parameters is discussed.

Effect of exogenous amino acids on the intracellular content of proline and other amino acids in Daucus carota cells.

The effect of tryptophan on the biosynthesis of proline has been investigated. Cells of Daucus carota grown in B5 medium supplemented with 5×10(-4)M tryptophan acquired the ability to grow in the presence of inhibitory concentrations of azetidine-2-carboxylic acid, an analog of proline. When trp was added to carrot cell cultures at sub-growth inhibiting concentrations, overproduction of intracellular free proline was observed. An increase was also observed for lys, his, ala, leu and phe. Likewise, the addition of asparagine, glutamic acid and phenylalanine to the medium stimulated the intracellular increase of free proline and other amino acids.

Mechanism of nitrofurantoin toxicity and oxidative stress in mitochondria.

5-Nitrofuran derivatives change the inner mitochondrial membrane permeability as indicated by the transmembrane potential, the rate of spontaneous K+ efflux and the basal respiratory rate: (a) at low concentrations nitrofurantoin prevents the increase of inner membrane permeability due to hydroperoxides or to diamide; (b) at higher concentrations or after longer times of incubation, nitrofurantoin enhances the membrane damage due to hydroperoxides or to diamide; the damage due Ca2+ plus Pi is enhanced by nitrofurantoin at all concentrations; (c) higher nitrofurantoin concentrations cause membrane damage independently of the presence of hydroperoxides or of diamide. The effect of nitrofurantoin is cancelled by the addition of free-radical scavengers. The above effects of nitrofurantoin are compatible with the observations of Mason and colleagues that nitrofurantoin is reduced by a NADPH nitroreductase to a nitro anion radical which can then undergo subsequent reactions, among which are (a) initiation of a free-radical reaction chain and (b) reduction of hydroperoxides and diamide.

Permeability of inner mitochondrial membrane and oxidative stress.

The mechanism of increase in the inner membrane permeability induced by Ca2+ plus Pi, diamide and hydroperoxides has been analyzed. (1) The permeability increase is antagonized by oligomycin and favoured by atractyloside. The promoting effect of atractyloside is strongly reduced if the mitochondria are simultaneously treated with oligomycin. (2) Addition of the free-radical scavenger, butylhydroxytoluene, results in a complete protection of the membrane with respect to the permeability increase. (3) Although membrane damage and depression of the GSH concentration are often associated, there is no direct correlation between extent of membrane damage and concentration of reduced glutathione. Abolition of the permeability increase by butylhydroxytoluene or by oligomycin is not accompanied by maintenance of a high GSH concentration in the presence of diamide or hydroperoxides. The membrane damage induced by Ca2+ plus Pi is not accompanied by a depression of the GSH concentration. (4) It is proposed that a variety of processes causing an increased permeability of the inner mitochondrial membrane merge into some ultimate common steps involving the action of oxygen radicals.

Production and Preliminary Characterization of Monoclonal Antibodies against Cationic Peanut Peroxidase.

Ten monoclonal antibodies (McAbs) have been produced against the cationic peroxidase from peanut suspension cell culture. Eight of these antibodies were found to be of the immunoglobulin (Ig)G(1) subclass and two were of IgA subclass. A combination of competitive enzyme-linked immunosorbent assay, Western blotting analysis, and direct antigen-binding assay revealed that the antibodies are directed against four different epitopes on the cationic peroxidase and the McAbs can be subdivided into four groups. Only group A inhibits peroxidase activity. Group B and D bind equally well to the native and the denatured form of cationic peroxidase, whereas the remaining McAbs react with more or less reduced affinity to the denatured antigen. Group C probably recognizes a conformation-dependent epitope. All the McAbs cross react weakly with the anionic peanut peroxidase, suggesting a structural nonidentity as well as some similarity between these two peroxidase isozymes. Cross reactivities of these McAbs with peroxidases of various plant species were also demonstrated.

Dihydrofolate reductase from Daucus carota cell suspension cultures: purification, molecular and kinetic characterization.

The purification of dihydrofolate reductase (5, 6, 7, 8 tetrahydrofolate: NADP(+) oxidoreductase, E.C.: 1.5.1.3) from Daucus carota to apparent homogeneity, is described. The enzyme is a soluble protein with a molecular weight of 183 000±2 500, composed of identical subunits of 58 400±1 000. The enzyme is only weakly recognized by antibodies against human DHFR. The carrot DHFR is characterized by a pH optimum of 5.9, Km values for dihydrofolate and NADPH of 3.7 µM and 2.2 µM, respectively and a turnover number of 4 750 or 1 500 when referring to the 183 K form or the 58 K monomer, respectively. Molecular and kinetic properties are remarkably different from those reported for the soybean enzyme. Sensitivity to methotrexate is similar to that of bacterial and mammalian enzymes while sensitivity to trimethoprim and dihydrotriazine is intermediate between the two groups of organisms.