Abstract: Species within the genus Populus include potential phytoextractors of heavy metal ions from contaminated soils, and genetic markers predictive of performance would be a useful tool for selection and breeding. Here, we have identified sequence variation within seven target and three nontarget genes among a set of 11 Populus spp. clones. Sequence variants were present in both the coding and noncoding regions; the former can potentially affect the functionality of the target genes. At the same time, the effect of exposure of the clones to cadmium ions on the morphology and the distribution of various metal ions was investigated by scanning electron microscopy microanalysis. A positive correlation was established between genetic variation, cadmium accumulation, and its bioconcentration in the root.
Abstract: Phytoremediation is a green, sustainable and promising solution to problems of environmental contamination. It entails the use of plants for uptake, sequestration, detoxification or volatilization of inorganic and organic pollutants from soils, water, sediments and possibly air. Phytoremediation was born from the observation that plants possessed physiological properties useful for environmental remediation. This was shortly followed by the application of breeding techniques and artificial selection to genetically improve some of the more promising and interesting species. Now, after nearly 20 years of research, transgenic plants for phytoremediation have been produced, but none have reached commercial existence. Three main approaches have been developed: (1) transformation with genes from other organisms (mammals, bacteria, etc.); (2) transformation with genes from other plant species; and (3) overexpression of genes from the same plant species. Many encouraging results have been reported, even though in some instances results have been contrary to expectations. This review will illustrate the main examples with a critical discussion of what we have learnt from them.
Abstract: Worldwide, there are many large areas moderately contaminated with heavy metals and/or organics that have not been remediated due to the high cost and technical drawbacks of currently available technologies. Methods with a good potential for coping with these limitations are emerging from phytoremediation techniques, using, for example, specific amendments and/or plants selected from various candidates proven in several investigations to be reasonably efficient in extracting heavy metals from soil or water, or in co-metabolizing organics with bacteria flourishing or inoculated in their rhizospheres. Populus and Salix spp., two genera belonging to the Salicaceae family, include genotypes that can be considered among the candidates for this phytoremediation approach. This review shows the recent improvements in analytical tools based on the identification of useful genetic diversity associated with classical growth, physiological and biochemical traits, and the importance of plant genotype selection for enhancing phytoremediation efficiency. Particularly interesting are studies on the application of the phytoremediation of heavy metals and of chlorinated organics, in which microorganisms selected for their degradation capabilities were bioaugmented in the rhizosphere of Salicaceae planted at a high density for biomass and bioenergy production.
Abstract: We have characterised genetically and phenotypically a T-DNA insertion mutant line of A. thaliana (L.) Heynh. selected for Cs resistance when germinating and growing on Cs concentrations up to 600microM, lethal for the wild type. Measures of concentration and localisation of Cs, K, and Ca have been conducted on plants grown in vivo also utilising synchrotron light-based techniques as micro-SRXF (synchrotron radiation induced X-ray micro-fluorescence) and micro-XANES (micro X-ray absorption near edge structure) spectroscopy. We report here an attempt to apply micro-XANES to investigate Cs speciation and to measure the Cs content of living plants. The results obtained with micro-SRXF and micro-XANES spectroscopy complemented the genetic and physiological analyses: a comparison between wild type and mutant plants led to the conclusion that in our case a single gene mutation impairs Cs uptake and translocation, K and Ca homeostasis and plant biomass production.
Abstract: This paper reviews aspects relevant to detection and quantification of genetically modified (GM) material within the feed/food chain. The GM crop regulatory framework at the international level is evaluated with reference to traceability and labelling. Current analytical methods for the detection, identification, and quantification of transgenic DNA in food and feed are reviewed. These methods include quantitative real-time PCR, multiplex PCR, and multiplex real-time PCR. Particular attention is paid to methods able to identify multiple GM events in a single reaction and to the development of microdevices and microsensors, though they have not been fully validated for application.
Abstract: One of the burning problems of our industrial society is the high consumption of water and the high demand for clean drinking water. Numerous approaches have been taken to reduce water consumption, but in the long run it seems only possible to recycle waste water into high quality water. It seems timely to discuss alternative water remediation technologies that are fit for industrial as well as less developed countries to ensure a high quality of drinking water throughout Europe.
Abstract: Molecular methodologies are increasingly being developed to assess the origin and authenticity of raw organic materials and processed food products. Here we describe the application of a microarray-based assay for single nucleotide polymorphisms (SNPs) identification in olive cultivars. The assay distinguishes alleles in a ligation detection reaction (LDR), with subsequent fluorescent detection by hybridization on a universal array (UA). The LDR-UA approach was used to detect 17 SNPs in olive genomic sequences previously amplified by PCR from fresh olive leaves. Genotype calls obtained with the LDR-UA were in full agreement with those determined by direct sequencing. The panel of 17 SNPs is sufficient to discriminate 49 olive varieties selected among the most widely cultivated for olive oil production in the Mediterranean area.
Abstract: DNA analysis with molecular markers has opened a shortcut toward a genomic comprehension of complex organisms. The availability of micro-DNA extraction methods, coupled with selective amplification of the smallest extracted fragments with molecular markers, could equally bring a breakthrough in food genomics: the identification of original components in food. Amplified fragment length polymorphisms (AFLPs) have been instrumental in plant genomics because they may allow rapid and reliable analysis of multiple and potentially polymorphic sites. Nevertheless, their direct application to the analysis of DNA extracted from food matrixes is complicated by the low quality of DNA extracted: its high degradation and the presence of inhibitors of enzymatic reactions. The conversion of an AFLP fragment to a robust and specific single-locus PCR-based marker, therefore, could extend the use of molecular markers to large-scale analysis of complex agro-food matrixes. In the present study is reported the development of sequence characterized amplified regions (SCARs) starting from AFLP profiles of monovarietal olive oils analyzed on agarose gel; one of these was used to identify differences among 56 olive cultivars. All the developed markers were purposefully amplified in olive oils to apply them to olive oil traceability.
Abstract: European walnut (Juglans regia) plants were grown in pots, on peat soil contaminated with lead (Pb), for four years. European walnut was chosen because it grows in Mediterranean climates, it yields a high biomass, and a fine quality wood. In the above ground parts Pb concentration was 1000 times lower than in roots: in 50 g roots there was 450 mg of Pb. Microanalysis of roots found in periderm more than 50% of the total root Pb. Pb L(III) EXAFS spectroscopy was performed on: root powder from Pb-exposed plants, Pb-impregnated cellulose and lignin. Comparison of plant material with lignin and cellulose helped to envisage a plant disposal strategy for Pb. This may consist in establishing links with large organic molecules, which are abundant constituents of cell walls. EXAFS spectroscopy evidenced the presence of Pb-O bindings within the ligno-cellulosic structure in roots. Lead was scantly conveyed to the shoots, giving to walnut plants an added asset in Pb phytostabilization.
Abstract: Two horizontal subsurface flow reed beds of 75 m(2) each, treating dairy parlor effluent and domestic sewage (about 6.5 m(3)/day), were set-up to determine the efficiency of this system in reducing the polluting load in an isolated mountain rural settlement.A total suspended solids value of about 0.70 g/l and chemical oxygen demand (COD) and biological oxygen demand (BOD(5)) values of about 1200 and 450 mg/l O(2), respectively, were characteristic of the influent waters. Removal of suspended solids and organic load constantly remained at levels above 90%, while those of the nutrients N and P were about 50% and 60%, respectively. The total number of coliform bacteria and Escherichia coli was reduced by more than 99%, and faecal streptococci by more than 98%. Nitrates, chlorides, sulfates, anionic and non-ionic surface-active agents and heavy metals were detected only in low concentrations. Concentration and localization of metals was also quantified in Phragmites tissues by microanalysis.Results demonstrated the use of reed beds as an appropriate treatment to reduce pollutants in wastewater from rural activities to values acceptable for discharge into surface waters.
Abstract: Heat stress is common in most cereal-growing areas of the world. In this paper, we summarize the current knowledge on the molecular and genetic basis of thermotolerance in vegetative and reproductive tissues of cereals. Significance of heat stress response and expression of heat shock proteins (HSPs) in thermotolerance of cereal yield and quality is discussed. Major avenues for increasing thermotolerance in cereals via conventional breeding or genetic modification are outlined.
Abstract: Three types of molecular markers have been compared for their utility in evaluating genetic diversity among cultivars of Hordeum vulgare. Restriction fragment length polymorphisms at 71 sites were scored with the aid of probes corresponding to stress-responsive genes from barley and wheat, coding for a low-molecular-weight heat shock protein, a dehydrin, an aldose reductase homolog, and a 18.9-kDa drought-induced protein of unknown function. Indexes of genetic diversity computed in the total sample and within groups of cultivars (two-rowed and six-rowed, winter and spring varieties) indicated high values of genetic differentiation ( F (ST) >15%). A second assessment of genetic diversity was performed by PCR amplification of genomic DNA using as primers 13 arbitrary oligonucleotides derived from sequences of the same stress-responsive genes. A high degree of polymorphism was uncovered using these markers also, but they yielded low values for F (ST) (<7%) among groups of cultivars. Finally, 15 different simple-sequence repeats (AC or AG) were amplified with primers based on unique flanking sequences. Levels of polymorphism and differentiation between groups of cultivars revealed by these markers were quite high. Ordination techniques applied to measures of genetic distance among cultivars demonstrated a remarkable ability of the RFLPs associated with stress-responsive genes to discriminate on the basis of growth habit. The correlation with production data for the cultivars in different environments was also significant. This "functional genomics" strategy was therefore as informative as the "structural genomics" (SSR-based) approach, but requires the analysis of fewer probes.
Abstract: A total of 568 new simple sequence repeat (SSR)-based markers for barley have been developed from a combination of database sequences and small insert genomic libraries enriched for a range of short simple sequence repeats. Analysis of the SSRs on 16 barley cultivars revealed variable levels of informativeness but no obvious correlation was found with SSR repeat length, motif type, or map position. Of the 568 SSRs developed, 242 were genetically mapped, 216 with 37 previously published SSRs in a single doubled-haploid population derived from the F(1) of an interspecific cross between the cultivar Lina and Hordeum spontaneum Canada Park and 26 SSRs in two other mapping populations. A total of 27 SSRs amplified multiple loci. Centromeric clustering of markers was observed in the main mapping population; however, the clustering severity was reduced in intraspecific crosses, supporting the notion that the observed marker distribution was largely a genetical effect. The mapped SSRs provide a framework for rapidly assigning chromosomal designations and polarity in future mapping programs in barley and a convenient alternative to RFLP for aligning information derived from different populations. A list of the 242 primer pairs that amplify mapped SSRs from total barley genomic DNA is presented.
Abstract: Simple sequence repeat (SSR)-based genetic markers are being actively developed for the majority of crop plant species. In barley, characterization of 290 dinucleotide repeat-containing clones from SSR-enriched libraries has revealed that a high percentage are associated with cereal retrotransposon-like and other dispersed repetitive elements. Associations found were with BARE-1, WIS2-1A, PREM1 and the dispersed repetitive element R173. Additional similarities between different SSR clones, which have no matches in DNA sequence databases, indicate that this phenomenon is probably widespread in the barley genome. Sequence homologies to the non-coding regions of several cereal genes were also explained by homology to mobile genetic elements. The SSRs found can therefore be classified into two types: (1) those with unique sequences on either flank, and (2) those which are intimately associated with retro-transposons and other dispersed repetitive elements. As the cereal genome is thought to consist largely of this type of DNA, some random association would be expected. However, the conserved positions of the SSRs, relative to repetitive elements, indicate that they have arisen non-randomly. Furthermore, this class of SSRs can be classified into three subtypes: (1) those which are positioned 3' of a transposable element with unique sequence on the other flank, (2) those positioned 5' of a transposable element, and (3) those which have arisen from an internal sequence and so have transposable element sequence on both flanks. The first appear to be analogous to the class of SSRs in mammalian systems which are associated with Alu elements and SINEs (short interspersed elements) and which have been postulated to arise following integration of an extended and polyadenylated retro-transcript into the host genome, followed by mutation of the poly(A) tract and expansion into an SSR. For the second, we postulate that a proto-SSR (A-rich sequence) has acted as a 'landing pad' for transposable element insertion (rather than being the result of insertion), while the third includes those which have evolved as a component of an active transposable element which has spread throughout the genome during bursts of transposition activity. The implications of these associations for genome and SSR evolution in barley are discussed.
Abstract: Environmental pollutants can have deleterious effects on living organisms. At high concentrations, or at high activities, they can cause acute toxicity damaging cells, tissues and organs. Chronic toxification, on the other hand, can also cause serious damage from bio-accumulation. Plants, as biological indicators, can measure both the actual and the potential effects of pollutants, when they are used to measure effects of heavy metals. We have applied a system of "molecular fingerprinting" based on PCR (RAPD: Random Amplified Polymorphic DNA) to the evaluation of the genotoxic effects of heavy metals in order to estimate the environmental risk connected with their potential mutagenic effects in the model plant Arabidopsis thaliana, ecotype Columbia. Genomic DNA was utilised for RAPD analysis using random primers (10-mers). DNA from plants exposed to heavy metals solution displayed polymorphic bands which were not detectable in DNA of unexposed plants. The enhanced formation of RAPD polymorphisms was also observed in DNA of plant exposed in situ to an industrial pollution source. The comparison between "unexposed" and "exposed" genomes show that RAPD analysis can be used to evaluate how the environmental pollutants modify the structure of DNA in living organisms.
Abstract: Plants of Arabidopsis thaliana pre-treated at 37 degrees C for 2 h can survive an otherwise lethal heat shock at 45 degrees C. Differential display reverse transcriptase-PCR (DDRT-PCR) was utilized to clone DNA fragments corresponding to mRNAs specifically expressed in conditions of induced thermotolerance or of expression of thermotolerance. One of these DDRT-PCR fragments enabled the isolation of a genomic clone pAt1.3EX, containing the sequence Athsp23.5, the gene for a low-molecular-weight (LMW) heat shock protein (HSP), AtHSP23.5. Athsp23.5 is low- or single-copy in the Arabidopsis genome and its open reading frame is interrupted by a 137 bp intron. Analysis of the sequence suggests AtHSP23.5 is targeted to the mitochondrion. The steady-state level of the AtHSP23.5 mRNA varied significantly according to the heat treatment, increasing on heat shock (transfer from 22 degrees C to 37 degrees C), with a further increase during expression of thermotolerance (transfer from 22 degrees C to 37 degrees C and then to 45 degrees C). Expression was low after an abrupt stress (from 22 degrees C to 45 degrees C). This behaviour was different from that observed for other LMW HSP mRNAs that were present at high level at 37 degrees C, but did not increase significantly in condition of expression of thermotolerance, and reached a considerable steady-state level also during the abrupt stress at 45 degrees C. The retrotranscription of AtHSP23.5 mRNA followed by amplification with two primers encompassing the intron allowed for the isolation of an almost full-length cDNA sequence. The sequence analysis of the two cDNAs obtained from condition 22 degrees C-->37 degrees C and condition 22 degrees C-->45 degrees C suggested that in both cases the intron had been correctly spliced. The importance of correct intron splicing in survival at high temperatures and the role of mitochondrial HSP in induction and expression of thermotolerance are discussed.
