[Show abstract][Hide abstract] ABSTRACT: We successfully combined ultrastructure and chloroplast DNA analysis for single specimens of Concentricystis isolated from Holocene sediment
by a palynology method based on filtration, progressive sieving and percoll-gradient sedimentation to concentrate fossil cells. We compared
our method with the traditional harsh chemical treatment commonly used to isolate fossil pollen and spores. With our method, the
cytoplasm of Concentricystis was sufficiently preserved to distinguish several cell structures by light and electron microscopy. DNA analysis
of Concentricystis involved PCR amplification using specific primers for a spacer region of the chloroplast genome. Significant homologies
were found with the Angiosperm chloroplast genome by BLAST DNA search for PCR product sequences.
� 2005 Elsevier Ltd. All rights reserved.
Journal of Archaeological Science 01/2006; DOI:10.1016/j.jas.2005.11.014 · 2.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] 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 Arlequin 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.
Scientia Horticulturae 02/2003; 97(3):379-388. DOI:10.1016/S0304-4238(02)00163-2 · 1.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: MADS-box genes are known to be important for the development of flowers and fruit. Four MADS-box cDNAs were cloned from grapevine flowers and unripe berries. When compared with MADS-box genes from Arabidopsis, two of the grape clones (VvMADS2 and VvMADS4) were related to the SEPALLATA (SEP) genes, a third (VvMADS3) showed homology to AGAMOUS-LIKE 6 and 13 (AGL6 and 13) and the fourth (VvMADS5) to AGL11. Expression of the grapevine MADS-box genes varied depending on the stage of inflorescence development, although all were expressed in fully developed flowers. VvMADS5 expression was restricted to the carpels and was found to be expressed in female but not male flowers. The other three MADS-box genes were expressed in the three inner whorls and in both male and female flowers. The expression of VvMADS4 was also detected at high levels during berry development, whereas VvMADS2 and VvMADS5 were detected at lower levels in berries. The gene expression patterns suggest that these genes have a role in regulating both grapevine flower and berry development.
[Show abstract][Hide abstract] ABSTRACT: A comparison between two recently developed, PCR based DNA marker technologies (amplified fragment length polymorphism, AFLP; inverse sequence- tagged repeat analysis, ISTR) was carried out in a group of 19 Vitis vinifera L. accessions, including 13 putative Sangiovese-related grapevines and 6 'coloured' ecotypes whose fruits are of importance for conferring intense red colour to the wine. A large amount of polymorphic DNA fragments was revealed by both molecular techniques: 8 different AFLP and 5 ISTR primer combinations generated 264 and 249 polymorphic markers, respectively. Similarity relationships among the accessions were described by cluster analysis. The AFLP analysis revealed the existence of a uniform group for the Sangiovese (SG) ecotypes showing a high degree of genetic relatedness for the members of this cultivar. Among the coloured ecotypes (CLR), variability was more evident. Only the so called Colorino americano ecotype significantly diverged from both groups. ISTR analysis confirmed the genetic dissimilarity of Colorino americano and the existence of the SG and CLR groups, but in addition detected a higher proportion of polymorphism among the Sangiovese accessions compared to AFLP analysis. Sangiovese forte and Saragiolo apparently differed from the other SG related grapevines in agreement with AFLP results. It is possible that the observed genetic dissimilarity between Sangiovese forte, Saragiolo and other SG-related types could be interpreted by the putative polyclonal origin of many grapevine cultivars, a concept which is generally accepted by the grapevine research community. Both AFLP and ISTR appear to represent innovative, efficient and sensitive molecular tools for investigating genetic diversity among Vitis vinifera ecotypes and for the eventual identification of clones.
[Show abstract][Hide abstract] ABSTRACT: Microsatellite (MS) VVMD21 (BOWERS et al. 1999) was taken as a model to explore the molecular basis of polymorphism in a panel of 6 grapevine accessions (Vitis vinifera L.), consisting of Sangiovese and Cabernet Sauvignon and 4 F 1 plants derived from crossing both vari-eties. The 12 alleles of both parents and the progeny were cloned and sequenced. The microsatellite repeat (AG) n>6 was found in each sequence, together with a poly-T rich region that showed irregularity. Furthermore, single nu-cleotide deletion or exchange (point mutations) were found in the microsatellite flanking regions. K e y w o r d s : MS polymorphism, DNA, sequence, Vitis vinifera.