Validation of a simplified small-scale DNA extraction protocol from wine by quantitative real-time PCR.

In the present study, we compared a simplified small-scale purification protocol to obtain DNA admixtures out of wine, with our large-scale published method. The extraction methods must provide DNA free of PCR inhibitors, that can interfere with DNA amplification. To evaluate the efficiency of grapevine's nuclear DNA extraction from wine, the new protocol was also compared in terms of purity and yield to the DNA obtained out of grapevine's (Vitis vinifera) leaf tissue, using a commercial kit. Two single-copy nuclear genes, nine-cis-epoxy carotenoid dioxygenase 2 (NCED2), and prefoldin subunit 5-like (PS5) were amplified in DNA extracted from wine and grapevine by real-time TaqMan PCR to determine the presence of inhibitors in relation to the diversity of starting biological matrix. This study showed that the small-scale, simpler method for extracting DNA from wine produced effective results in terms of inhibitor presence and purity. Furthermore, even though the initial biological matrix was more complicated, the grapevine nuclear DNA that was removed from wine was qualitatively equivalent to the DNA that was isolated from the leaves. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-03992-x.

Cadmium tolerance and hyperaccumulation in plants - A proteomic perspective of phytoremediation.

Cadmium (Cd) is a major environmental pollutant and poses a risk of transfer into the food chain through contaminated plants. Mechanisms underlying Cd tolerance and hyperaccumulation in plants are not fully understood. Proteomics-based approaches facilitate an in-depth understanding of plant responses to Cd stress at the systemic level by identifying Cd-inducible differentially abundant proteins (DAPs). In this review, we summarize studies related to proteomic changes associated with Cd-tolerance mechanisms in Cd-tolerant crops and Cd-hyperaccumulating plants, especially the similarities and differences across plant species. The enhanced DAPs identified through proteomic studies can be potential targets for developing Cd-hyperaccumulators to remediate Cd-contaminated environments and Cd-tolerant crops with low Cd content in the edible organs. This is of great significance for ensuring the food security of an exponentially growing global population. Finally, we discuss the methodological drawbacks in current proteomic studies and propose that better protocols and advanced techniques should be utilized to further strengthen the reliability and applicability of future Cd-stress-related studies in plants. This review provides insights into the improvement of phytoremediation efficiency and an in-depth study of the molecular mechanisms of Cd enrichment in plants.

Genetic Engineering of Starch Biosynthesis in Maize Seeds for Efficient Enzymatic Digestion of Starch during Bioethanol Production.

Maize accumulates large amounts of starch in seeds which have been used as food for human and animals. Maize starch is an importantly industrial raw material for bioethanol production. One critical step in bioethanol production is degrading starch to oligosaccharides and glucose by α-amylase and glucoamylase. This step usually requires high temperature and additional equipment, leading to an increased production cost. Currently, there remains a lack of specially designed maize cultivars with optimized starch (amylose and amylopectin) compositions for bioethanol production. We discussed the features of starch granules suitable for efficient enzymatic digestion. Thus far, great advances have been made in molecular characterization of the key proteins involved in starch metabolism in maize seeds. The review explores how these proteins affect starch metabolism pathway, especially in controlling the composition, size and features of starch. We highlight the roles of key enzymes in controlling amylose/amylopectin ratio and granules architecture. Based on current technological process of bioethanol production using maize starch, we propose that several key enzymes can be modified in abundance or activities via genetic engineering to synthesize easily degraded starch granules in maize seeds. The review provides a clue for developing special maize cultivars as raw material in the bioethanol industry.

Sterols and Sphingolipids as New Players in Cell Wall Building and Apical Growth of Nicotiana tabacum L. Pollen Tubes.

Pollen tubes are tip-growing cells that create safe routes to convey sperm cells to the embryo sac for double fertilization. Recent studies have purified and biochemically characterized detergent-insoluble membranes from tobacco pollen tubes. These microdomains, called lipid rafts, are rich in sterols and sphingolipids and are involved in cell polarization in organisms evolutionarily distant, such as fungi and mammals. The presence of actin in tobacco pollen tube detergent-insoluble membranes and the preferential distribution of these domains on the apical plasma membrane encouraged us to formulate the intriguing hypothesis that sterols and sphingolipids could be a "trait d'union" between actin dynamics and polarized secretion at the tip. To unravel the role of sterols and sphingolipids in tobacco pollen tube growth, we used squalestatin and myriocin, inhibitors of sterol and sphingolipid biosynthesis, respectively, to determine whether lipid modifications affect actin fringe morphology and dynamics, leading to changes in clear zone organization and cell wall deposition, thus suggesting a role played by these lipids in successful fertilization.

Protein Analysis of Pollen Tubes after the Treatments of Membrane Trafficking Inhibitors Gains Insights on Molecular Mechanism Underlying Pollen Tube Polar Growth.

Pollen tube elongation is characterized by a highly-polarized tip growth process dependent on an efficient vesicular transport system and largely mobilized by actin cytoskeleton. Pollen tubes are an ideal model system to study exocytosis, endocytosis, membrane recycling, and signaling network coordinating cellular processes, structural organization and vesicular trafficking activities required for tip growth. Proteomic analysis was applied to identify Nicotiana tabacum Differentially Abundant Proteins (DAPs) after in vitro pollen tube treatment with membrane trafficking inhibitors Brefeldin A, Ikarugamycin and Wortmannin. Among roughly 360 proteins separated in two-dimensional gel electrophoresis, a total of 40 spots visibly changing between treated and control samples were identified by MALDI-TOF MS and LC-ESI-MS/MS analysis. The identified proteins were classified according to biological processes, and most proteins were related to pollen tube energy metabolism, including ammino acid synthesis and lipid metabolism, structural features of pollen tube growth as well modification and actin cytoskeleton organization, stress response, and protein degradation. In-depth analysis of proteins corresponding to energy-related pathways revealed the male gametophyte to be a reliable model of energy reservoir and dynamics.

How to integrate wet lab and bioinformatics procedures for wine DNA admixture analysis and compositional profiling: Case studies and perspectives.

The varietal authentication of wines is fundamental for assessing wine quality, and it is part of its compositional profiling. The availability of historical, cultural and chemical composition information is extremely important for quality evaluation. DNA-based techniques are a powerful tool for proving the varietal composition of a wine. SSR-amplification of genomic residual Vitis vinifera DNA, namely Wine DNA Fingerprinting (WDF) is able to produce strong, analytical evidence concerning the monovarietal nature of a wine, and for blended wines by generating the probability of the presence/absence of a certain variety, all in association with a dedicated bioinformatics elaboration of genotypes associated with possible varietal candidates. Together with WDF we could exploit Bioinformatics techniques, due to the number of grape genomes grown. In this paper, the use of WDF and the development of a bioinformatics tool for allelic data validation, retrieved from the amplification of 7 to 10 SSRs markers in the Vitis vinifera genome, are reported. The wines were chosen based on increasing complexity; from monovarietal, experimental ones, to commercial monovarietals, to blended commercial wines. The results demonstrate that WDF, after calculation of different distance matrices and Neighbor-Joining input data, followed by Principal Component Analysis (PCA) can effectively describe the varietal nature of wines. In the unknown blended wines the WDF profiles were compared to possible varietal candidates (Merlot, Pinot Noir, Cabernet Sauvignon and Zinfandel), and the output graphs show the most probable varieties used in the blend as closeness to the tested wine. This pioneering work should be meant as to favor in perspective the multidisciplinary building-up of on-line databanks and bioinformatics toolkits on wine. The paper concludes with a discussion on an integrated decision support system based on bioinformatics, chemistry and cultural data to assess wine quality.

Microtubules play a role in trafficking prevacuolar compartments to vacuoles in tobacco pollen tubes.

Fine regulation of exocytosis and endocytosis plays a basic role in pollen tube growth. Excess plasma membrane secreted during pollen tube elongation is known to be retrieved by endocytosis and partially reused in secretory pathways through the Golgi apparatus. Dissection of endocytosis has enabled distinct degradation pathways to be identified in tobacco pollen tubes and has shown that microtubules influence the transport of plasma membrane internalized in the tip region to vacuoles. Here, we used different drugs affecting the polymerization state of microtubules together with SYP21, a marker of prevacuolar compartments, to characterize trafficking of prevacuolar compartments in Nicotiana tabacum pollen tubes. Ultrastructural and biochemical analysis showed that microtubules bind SYP21-positive microsomes. Transient transformation of pollen tubes with LAT52-YFP-SYP21 revealed that microtubules play a key role in the delivery of prevacuolar compartments to tubular vacuoles.

Distribution and Characterization of the Vitis vinifera L. subsp sylvestris in Southern Tuscany.

BACKGROUND: The Eurasian grapevine is the most widespread fruit crop in the world. Numerous studies have focused on clarifying the events of domestication and the geographical areas in which they occurred. OBJECTIVES: In order to add upon information on the process of grapevine domestication, the distribution and genetic diversity of a small, wild population localized in Poggio Ghiaccio Forte, an archaeological site in Maremma (Southern Tuscany), were assessed. In a preliminary survey the wild forms of Vitis vinifera L. were identified based on morphological traits. METHOD: Fourty-two accessions of Vitis vinifera ssp. sylvestris were collected near the Albegna river and its secondary conduits. As a control, four non-vinifera grapevines (Vitis berlandieri, Vitis riparia Fabre, rupestris Constantia, rupestris H. Goethe) and ten varieties of Vitis vinifera ssp. sativa characterizing the Tuscan grapevine germplasm (Sangiovese, Ciliegiolo, Aleatico, Ansonica, Canaiolo Nero, Trebbiano Toscano, Barsaglina, Malvasia Lunga, Moscato Bianco, Abrusco) were added to the wild population. All fifty-six vines were treated as one population and tested by 10 SSR-based genotyping. RESULTS: According to SSR analysis, the wild population seems to be characterized by a systematic reduction of observed compared to expected heterozygosity due to the tendency of inbreeding and genetic trait fixation. There are a lot of registered patents about different applications involving Vitis vinifera mostly relating to disease resistance, grapevine fitness and novel combinations of antioxidants useful in therapeutic, foodstuff and cosmetic fields.

Archaeobotanical reconstructions of vegetation and report of mummified apple seeds found in the cellar of a first-century Roman villa on Elba Island.

In the late Roman Republic period (2nd-1st century BC), in the area of San Giovanni on Elba Island, previously subject to intense extraction of iron ore, a rustic villa was established by Marco Valerio Messalla, a supreme Roman magistrate. The foundations of the walls were discovered and excavated by an archaeological mission. Palaeobotanical analysis of a set of stratigraphic layers was performed. Palynological slides showed remains of palynomorphic and non-pollen objects, while data combined with anthracological investigations confirmed the hypothesis that in the 1st century AD the villa was destroyed by a fire that created a compact crust under which were discovered four broken Roman amphorae containing about five hundred apple seeds. Comparisons of archaeological and fresh seeds from reference collections showed discontinuous morphology except for one group of archaeological samples. DNA was isolated from seeds that had well-preserved embryos in all groups. DNA extracts from archaeological, wild and modern domestic seeds (controls) were amplified by PCR and tested with SSR molecular markers, followed by genome analysis.

Characterisation of detergent-insoluble membranes in pollen tubes of Nicotiana tabacum (L.).

Pollen tubes are the vehicle for sperm cell delivery to the embryo sac during fertilisation of Angiosperms. They provide an intriguing model for unravelling mechanisms of growing to extremes. The asymmetric distribution of lipids and proteins in the pollen tube plasma membrane modulates ion fluxes and actin dynamics and is maintained by a delicate equilibrium between exocytosis and endocytosis. The structural constraints regulating polarised secretion and asymmetric protein distribution on the plasma membrane are mostly unknown. To address this problem, we investigated whether ordered membrane microdomains, namely membrane rafts, might contribute to sperm cell delivery. Detergent insoluble membranes, rich in sterols and sphingolipids, were isolated from tobacco pollen tubes. MALDI TOF/MS analysis revealed that actin, prohibitins and proteins involved in methylation reactions and in phosphoinositide pattern regulation are specifically present in pollen tube detergent insoluble membranes. Tubulins, voltage-dependent anion channels and proteins involved in membrane trafficking and signalling were also present. This paper reports the first evidence of membrane rafts in Angiosperm pollen tubes, opening new perspectives on the coordination of signal transduction, cytoskeleton dynamics and polarised secretion.

Universal sample preparation method integrating trichloroacetic acid/acetone precipitation with phenol extraction for crop proteomic analysis.

Crop plants contain large amounts of secondary compounds that interfere with protein extraction and gel-based proteomic analysis. Thus, a protein extraction protocol that can be easily applied to various crop materials with minimal optimization is essential. Here we describe a universal protocol for total protein extraction involving trichloroacetic acid (TCA)/acetone precipitation followed by SDS and phenol extraction. Through SDS extraction, the proteins precipitated by the TCA/acetone treatment can be fully resolubilized and then further purified by phenol extraction. This protocol combines TCA/acetone precipitation, which aggressively removes nonprotein compounds, and phenol extraction, which selectively dissolves proteins, resulting in effective purification of proteins from crop tissues. This protocol can also produce high-quality protein preparations from various recalcitrant tissues, and therefore it has a wide range of applications in crop proteomic analysis. Designed to run on a small scale, this protocol can be completed within 5 h.

Genetic differentiation between cinta senese and commercial pig breeds using microsatellite

Background: Cinta Senese (CS) is an autochthonous Tuscan breed, which risked extinction since the ‘60s. Results: Monitoring the genetic variability of the actual population by use of DNA molecular markers is essential to address a correct breeding policy, finalized to obtain the race preservation and its fitness in the future. 17 SSRs autosomal markers and 1 associated to the X chromosome were used to genotype 86 individuals belonging to the CS and 12 belonging to two main white races Landrace (L), Large White (LW) and crosses between LW and L and L and CS widespread in Tuscany and used in the recent past to obtain hybrids with the CS. Conclusions: A dendrogram of similarity measures the relative genetic distance between individuals in the population. Data show that CS pigs have a distinct genotype from L, LW, LW x L and L x CS.

Application of a suite of biomarkers in Posidonia oceanica (L.) delile to assess the ecotoxicological impact on the coastal environment.

The main aim of this study was to develop and validate a suite of sensitive responses (biomarkers) for monitoring conservation status and ecotoxicological impact in Posidonia oceanica meadows. Analytical methods were developed for NADPH cytochrome c reductase, ethoxycoumarin-o-deethylase (ECOD), guaiacol peroxidase (GPOX) and superoxide dismutase (SOD) assays. A preliminary proteomic approach using 2-D electrophoresis was also proposed as a biomarker. These techniques were initially tested on samples of posidonia exposed experimentally to various contaminants. Once validated, this approach was applied to posidonia in a field study. Samples of the seagrass were collected at four sites with potentially different environmental impact along the northern Tyrrhenian coast. The results showed that reductase activity was significantly induced in the various sampling areas with respect to the reference site. GPOX and SOD showed a similar trend; the highest activities were found in samples collected off a chlor-alkali plant and near a river estuary. Analysis of trace elements, polycyclic aromatic hydrocarbons (PAHs) and organochlorine compounds (OCs) in posidonia leaves showed differences between sites. A significant correlation was found between Hg concentrations and GPOX activity and between Cr, Al and As concentrations and reductase activity. The results validated these biomarkers in posidonia for the assessment of ecotoxicological impact on the coastal ecosystem.

A universal and rapid protocol for protein extraction from recalcitrant plant tissues for proteomic analysis.

A simple and universally applicable protocol for extracting high-quality proteins from recalcitrant plant tissues is described. We have used the protocol with no modification, for a wide range of leaves and fruits. In all cases, this protocol allows to obtain good electrophoretic separation of proteins. As the protocol is rapid, universal, and compatible with silver staining, it could be used for routine protein extraction from recalcitrant plant tissues for proteomic analysis.

A methionine synthase homolog is associated with secretory vesicles in tobacco pollen tubes.

Seven isoforms of 85 kDa polypeptides (p85) were identified as methionine synthase (MetE) homologs by partial aminoacid sequencing in tobacco pollen tube extracts. Immunocytochemistry data showed a localization of the antigen on the surface of tip-focussed post-Golgi secretory vesicles (SVs), that appear to be partially associated with microtubules (Mts). The chemical dissection of pollen tube high speed supernatant (HSS) showed that two distinct pools of MetE are present in pollen tubes, one being the more acidic isoforms sedimenting at 15S and the remaining at 4S after zonal centrifugation through a sucrose density gradient. The identification of the MetE within the pollen tube and its possible participation as methyl donor in a wide range of metabolic reactions, makes it a good subject for studies on pollen tube growth regulation.

Post-translational modifications of alpha-tubulin in Zea mays L are highly tissue specific.

To further understand post-translational modifications (PTMs) of plant alpha-tubulin, post-translationally modified alpha-tubulin isoforms from selected tissues of Zea mays L. were examined using two-dimensional electrophoresis and immunoblotting. Except for polyglycylated tubulin, tyrosinated, detyrosinated, acetylated and polyglutamylated alpha-tubulin isoforms were all present in maize tissues. Tyrosinated alpha-tubulin was the predominant variant in all cases, with isoforms alpha1-alpha4 (alpha5) being the most common components. Leaves exhibited a striking difference in PTM patterns of alpha-tubulin isoforms compared to other tissues examined. In leaves, several major specific isoforms were highly modified by detyrosination, acetylation and polyglutamylation. In pollen and anthers, only the most abundant isoform alpha3 was acetylated to an appreciable extent, and no acetylated isoform was found in roots. Similarly, in pollen, anthers and roots, only alpha3 was appreciably polyglutamylated. Additionally, a detyrosinated isoform alpha6 was present in anthers and in leaves, while the tyrosinated isoform alpha6 seemed to be pollen specific. These results indicate that certain types of PTM of plant alpha-tubulin preferentially occur in a tissue-specific way.

Protein extraction for two-dimensional electrophoresis from olive leaf, a plant tissue containing high levels of interfering compounds.

The purpose of this research is to establish a routine procedure for the application of proteomic analysis to olive tree. Olive leaf tissue is notoriously recalcitrant to common protein extraction methods due to high levels of interfering compounds. We developed a protocol for isolating proteins suitable for two-dimensional electrophoresis (2-DE) from olive leaf. The remarkable characteristics of the protocol include: (i) additional grinding dry acetone powder of leaf tissue to a finer extent, (ii) after extensive organic solvent washes to remove pigments, lipids etc., using aqueous tricholoroacetic acid washes to remove water-soluble contaminants, and (iii) phenol extraction of proteins in the presence of sodium dodecyl sulfate. The final protein preparation is free of interfering compounds based on its well-resolved 2-DE patterns. The protocol can be completed within 3 h, and protein yield is approximately 2.49 mg.g(-1) of aged leaf. We also evaluated the protocol by immunoblotting with anti-tyrosinate alpha-tubulin antibody. To our knowledge, this is the first time that a protocol for protein extraction from olive leaf appears to give satisfactory and reproducible results. The protocol is expected to be applicable to other recalcitrant plant tissues and could be of interest to laboratories involved in plant proteomics.