Monica Scali

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.

DNA fingerprinting and genetic relatedness among cultivated varieties of Olea europaea L. estimated by AFLP analysis

Abstract Amplified fragment length polymorphism (AFLP) analysis was used to evaluate the genetic biodiversity and variability present in some Italian varieties of cultivated olive. A group of 12 genotypes belonging to three varieties was screened using six different AFLP primer combinations. A total of 274 loci (59.8% of which were polymorphic) were investigated. The number of polymorphic loci detected by single primer combination for each variety was calculated. AFLP banding patterns were transformed into binary data of presence–absence and matrices were processed with NTSYS- pc and A rlequin software programmes. Similarity relationships were described graphically by a dendrogram which clustered accessions of the same cultivar. Analysis of molecular variance (AMOVA) revealed greater variation among cultivars than within them, but significant intra-cultivar differentiation was found. For the varieties analysed, the data revealed significant genetic diversity in the cultivated olive tree, despite the fact that they come from a limited geographical area. The DNA fingerprinting technique used was confirmed to be a reproducible and sensitive tool for the study of population genetics of olive trees. The high genomic polymorphism observed suggests a more complex genetic population structure than the conventional variety or cultivar level. The present study confirms the importance of considering the degree of genetic relatedness and variability within populations during clone and variety selection programmes.

DISTRIBUTION OF MICROTUBULES DURING THE GROWTH OF TOBACCO POLLEN TUBES

Summary— The distribution of microtubules was investigated in Nicotiana tabacum pollen tubes at different stages of tube growth by immunofluorescence microscopy. Using specific antibodies, the presence of microtubules consisting of different tubulin isoforms was tested. α‐, β‐ and tyrosinated α‐tubulin were present within the tube, whereas the acetylated form was lacking. The presence of tubulin subunits in pollen tube extracts was also investigated by immunoblotting analyses. The use of a confocal laser scanning microscope integrated with computer‐assisted imaging, allowed a detailed visualization of the microtubule distribution and organization. Cytoplasmic microtubules organized as short bundles with various orientations were detected at the apex of long tubes.

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

To further understand post-translational modifications (PTMs) of plant α-tubulin, post-translationally modified α-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 α-tubulin isoforms were all present in maize tissues. Tyrosinated α-tubulin was the predominant variant in all cases, with isoforms α1–α4 (α5) being the most common components. Leaves exhibited a striking difference in PTM patterns of α-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 α3 was acetylated to an appreciable extent, and no acetylated isoform was found in roots. Similarly, in pollen, anthers and roots, only α3 was appreciably polyglutamylated. Additionally, a detyrosinated isoform α6 was present in anthers and in leaves, while the tyrosinated isoform α6 seemed to be pollen specific. These results indicate that certain types of PTM of plant α-tubulin preferentially occur in a tissue-specific way.

Confocal image analysis of spatial variations in immunocytochemically identified calmodulin during pollen hydration, germination and pollen tube tip growth in Nicotiana tabacum L.

Using monoclonal anti-calmodulin antibodies in conjunction with confocal scanning laser microscopy we have analysed the spatial variations in the distribution pattern of calmodulin (CaM) during the sequential events of pollen hydration, germination and tube growth in Nicotiana tabacum. These immunocytochemical observations have been complemented by immunochemical studies wherein the anti-calmodulin antibody raised against pea CaM recognises a polypeptide of c. 18 kDa in the pollen extracts. Digitisation of confocally acquired optical sections of immunofluorescence images reveals that in hydrated pollen a high level of CaM is consistently present in the region of the germinal apertures. Subsequently, with the onset of germination a high CaM concentration was found associated with the plasma membrane of the germination bubble and in the cytoplasm in its vicinity, while in the vegetative cytoplasm a weak diffuse and intense punctate signal was registered. CaM immunostain was also detected in association with the plasma membrane of the tube tips in both short and long pollen tubes. Furthermore, the cytosol of the tubes invariably manifested an apically focused CaM gradient. We were, however, unable to detect any vacuolar association of CaM in the older regions of the pollen tubes. Although punctate immunostain was obvious across the pollen tube numerous punctate structures were invariably present in the extreme tip. The possible implications of these findings in development of cell polarity, polarised growth, maintenance of calcium homeostasis and CaM interactions with other mechanochemical motor proteins in effecting propulsion of organelles during pollen hydration, germination and pollen tube growth are discussed.

Genomic variability in Vitis vinifera L. “Sangiovese” assessed by microsatellite and non-radioactive AFLP test

EJB Electronic Journal of Biotechnology ISSN: 0717-3458 Vol.5 No.1, Issue of April 15, 2002© 2002 by Universidad Catolica de Valparaiso -- Chile Received July 26, 2001 / Accepted March 11, 2002

Proteomic identification of differentially expressed proteins in mature and germinated maize pollen

The identification of proteins involved in pollen germination and tube growth is important for fundamental studies of fertility and reproduction in flowering plants. We used 2-DE and MALDI-TOF-MS to identify differentially expressed proteins in mature (P0) and 1-h germinated (P1) maize pollen. Among about 470 proteins separated in 2D gels, the abundances of 26 protein spots changed (up- or down-regulation) between P0 and P1. The 13 up-regulated protein spots were mainly involved in tube wall modification, actin cytoskeleton organization, energy metabolism, signaling, protein folding and degradation. In particular, pectin methylesterase, inorganic pyrophosphatase, glucose-1-phosphate uridylyltransferase and rab GDP dissociation inhibitor α are highly up-regulated, suggesting their potential role in pollen tube growth. The down-regulated 13 protein spots mainly include defense-related proteins, pollen allergens and some metabolic enzymes. This study would contribute to the understanding of the changes in protein expression associated with pollen tube development.

Proteomic analysis of β-1,3-glucanase in grape berry tissues

Grape berries are considered recalcitrant materials in proteomic analysis, because berry tissues contain large amounts of secondary metabolites, especially phenolic compounds, which severely interfere with protein extraction and electrophoresis separation. We report hereby a PVPP/TCA-based protein extraction protocol for grape berries. Phenolic compounds in berry extracts were removed with repeated PVPP cleanups, and proteins were recovered with TCA precipitation. Protein resolution in 2-D gels was gradually improved with the increase of PVPP cleanup steps. By the protocol, about 760 protein spots of berry tissues were clearly resolved in 2-D gels with CBB staining. This protocol was also used to analyze β-1,3-glucanase (EC 3.2.1.39) in berry tissues. An anti-synthetic peptide antibody was prepared against 15 amino acid sequence residing on the surface of β-1,3-glucanase molecule. It detected two major spots in 2-D blots of berry extracts. The spots were identified by MALDI-TOF analysis as β-1,3-glucanase. The present study validates that β-1,3-glucanase is present in higher abundance in berry skins than in pulps, and in red berries than in white berries. Therefore, β-1,3-glucanase displays a tissue-specific expression. The preferential accumulation of β-1,3-glucanase in skins may be relevant to berry ripening.

DNA Extracted with Optimized Protocols Can Be Genotyped to Reconstruct the Varietal Composition of Monovarietal Wines

A new DNA extraction protocol has been established to reconstruct the genetic identity of grapevine cultivars used for the production of experimental and commercial monovarietal wines. Seven wines originating from Merlot, Pinot noir, Zinfandel, Riesling, Sauvignon blanc, Sangiovese, and Alicante were identified by simple sequence repeat (SSR) analysis. The average quantity of Vitis vinifera DNA that can be extracted from wine, estimated by RT-PCR, was between 2,199 and 87,995 gc/mL. This is the first study that reports source vine identity reconstructed with a significant statistical correspondence (PI ≤ 10−6) in both experimental and commercial wines.

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

ABSTRACT 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.