http://http://dspace.unitus.it:80 The DSpace digital repository system captures, stores, indexes, preserves, and distributes digital research material. Thu, 23 May 2013 02:52:58 GMT 2013-05-23T02:52:58Z http://hdl.handle.net/2067/1727 Title: The ‘Evergrowing’ genotype of Corylus avellana is expressed in the offspring of ‘Tonda Gentile Romana’, ‘Nocchione’ and ‘Tonda di Giffoni’ Authors: Catarcione, Giulio; Vittori, Doriano; Ciaffi, Mario; Rugini, Eddo; De Pace, Ciro Abstract: The hazelnut (Corylus avellana L.) ‘evergrowing’ phenotype (EVG) fails both to cease growth and to enter dormancy under the dormancy-inducing (i.e., short days) conditions suitable for the wild type. The EVG phenotype is expressed by the homozygous genotype for the recessive allele EVG-d. Description: L'articolo é disponibile sul sito dell'editore: http://www.ishs.org Wed, 31 Dec 2008 23:00:00 GMT http://hdl.handle.net/2067/1727 2008-12-31T23:00:00Z http://hdl.handle.net/2067/1616 Title: Pattern of variation for seed size traits and molecular markers in Italian germplasm of Phaseolus coccineus L. using different molecular markers. Authors: Acampora, Andrea; Ciaffi, Mario; De Pace, Ciro; Paolacci, Anna Rita; Tanzarella, Oronzo A. Abstract: Variation in Italian germplasm of Phaseolus coccineus L. was assessed for seed traits and molecular markers. A total of 130 seeds and seedlings, five for each of 21 Italian landraces, an Italian commercial cultivar and four Mesoamerican landraces of P. coccineus, were analysed using seven selected PCR markers: three RAPDs, two ISSRs and two ETs. Seed weight of the Mesoamerican landraces was £1 g, whereas that of the Italian landraces varied from 1 g to 2.5 g and was related to their origin. Oval shape was more frequent, with round shape observed only in Mesoamerican landraces. Three seed coat colours were observed: white, violet mottled or spotted black and buff spotted brown, also this trait was related to the origin. The level of polymorphism detected by molecular markers was low but with significant discriminant power. ISSRs were the most effective markers prone to unravel molecular polymorphism. The within accession component of variation exceeded that among accessions, as expected for an allogamous species. However correct classification of the individuals was achieved performing either discriminant analysis of the seed phenotypic traits or cluster analysis of seedling similarity measure based on the whole banding patterns obtained by the three marker types. Our data suggest that the Italian farmers, starting with ancestral Mesoamerican runner bean introductions in Europe, bred their own landraces through selection for seed size and seed coat colour, but occasional gene flow maintained variability within landraces bred by different farmers in the same Italian Region. Selection favored molecular and seed trait uniformity within several landraces making them suitable for certification. Description: L'articolo è disponibile sul sito dell'editore: http://www.springerlink.com Sun, 31 Dec 2006 23:00:00 GMT http://hdl.handle.net/2067/1616 2006-12-31T23:00:00Z http://hdl.handle.net/2067/1613 Title: Gene expression induced by chronic ozone in the Mediterranean shrub Phillyrea latifolia: analylis cDNA-AFLP. Authors: Paolacci, Anna Rita; Miraldi, Cristiano; Tanzarella, Oronzo A.; Badiani, Maurizio; Porceddu, Enrico; Nali, Cristina; Lorenzini, Giacomo; Ciaffi, Mario Abstract: Seedlings of Phillyrea latifolia L., a Mediterranean shrub, were exposed for 90 days to 110 nl l –1 ozone (O3). Comparison of the cDNA-amplified fragment length polymorphism (cDNA-AFLP) patterns for treated and control plants allowed the identification and cloning of 88 differential sequences induced by O3. The differential expression of 67 cloned sequenceswas further confirmed byRT-PCR. The functions of 36 cloned sequences, corresponding to seven of the twelve gene functional classes of Arabidopsis, were presumed on the basis of their homology with characterized gene sequences. Ozone induction of genes homologous to 24 of the clones has been reported in other plant species, whereas the induction of the 12 remaining sequences has not been observed before. Ozone activation of these newly identified genes could be a result of the chronic exposure to low O3 concentration, because in most previous studies, acute treatments, involving high O3 dosages, were applied. Possible roles of the cloned sequences in the response of P. latifolia to O3 and other causes of oxidative stress are discussed. Sun, 31 Dec 2006 23:00:00 GMT http://hdl.handle.net/2067/1613 2006-12-31T23:00:00Z http://hdl.handle.net/2067/1619 Title: The PDI (Protein Disulfide Isomerase) gene family in wheat. Authors: d'Aloisio, Elisa; Tanzarella, Oronzo A.; Dhanapal, Arun Prabhu; Porceddu, Enrico; Ciaffi, Mario Abstract: The PDI (Protein Disulfide Isomerase) gene family includes several members whose products are responsible for diversified metabolic functions. PDI and PDI-like proteins differ for number and position of thioredoxin-like (TRX-like) active (a type) and inactive (b type) domains, for presence/absence of other domains and of the KDEL signal of retention in the endoplasmic reticulum (ER). The phylogenetic analysis of typical PDI and PDI-like protein sequences resolved them into 10 groups (1), 5 of them (I-V) had 2 TRX-like active domains, whereas the remaining ones owned only a single TRX-like active domain (VI-VIII, QSOX and APRL). In particular, QRX and APRL were not included in this study due to their putative non-isomerase enzymatic activities encoded by an additional domain. The aim of the present research was the study of the complexity and diversity of the PDI gene family in wheat, with particular focus on the genes encoding PDIlike proteins structurally similar to TaPDIL1-1 (group I), the first identified and best characterized member of the PDI family, also named typical PDI. The most important function of typical PDI is the formation and isomerization of disulfide bonds during protein folding, which are accomplished by its two active TRX-like sites sharing the characteristic tetrapeptide –CGHC-. Several studies of molecular characterization, expression analysis and cell localisation in rice and maize have suggested the involvement of typical PDI in the assembly and deposition of storage proteins in these species (2, 3, 4). The characterization and chromosome location of the three homoeologous gene sequences encoding typical PDI and of their promoter sequences have been reported previously (5). Mon, 31 Dec 2007 23:00:00 GMT http://hdl.handle.net/2067/1619 2007-12-31T23:00:00Z http://hdl.handle.net/2067/1609 Title: The Protein Disulfide Isomerase gene family in bread wheat (T. aestivum L.) Authors: d'Aloisio, Elisa; Paolacci, Anna Rita; Dhanapal, Arun Prabhu; Tanzarella, Oronzo A.; Porceddu, Enrico; Ciaffi, Mario Abstract: Background: The Protein Disulfide Isomerase (PDI) gene family encodes several PDI and PDI-like proteins containing thioredoxin domains and controlling diversified metabolic functions, including disulfide bond formation and isomerisation during protein folding. Genomic, cDNA and promoter sequences of the three homoeologous wheat genes encoding the "typical" PDI had been cloned and characterized in a previous work. The purpose of present research was the cloning and characterization of the complete set of genes encoding PDI and PDI like proteins in bread wheat (Triticum aestivum cv Chinese Spring) and the comparison of their sequence, structure and expression with homologous genes from other plant species. Results: Eight new non-homoeologous wheat genes were cloned and characterized. The nine PDI and PDI-like sequences of wheat were located in chromosome regions syntenic to those in rice and assigned to eight plant phylogenetic groups. The nine wheat genes differed in their sequences, genomic organization as well as in the domain composition and architecture of their deduced proteins; conversely each of them showed high structural conservation with genes from other plant species in the same phylogenetic group. The extensive quantitative RT-PCR analysis of the nine genes in a set of 23 wheat samples, including tissues and developmental stages, showed their constitutive, even though highly variable expression. Conclusions: The nine wheat genes showed high diversity, while the members of each phylogenetic group were highly conserved even between taxonomically distant plant species like the moss Physcomitrella patens. Although constitutively expressed the nine wheat genes were characterized by different expression profiles reflecting their different genomic organization, protein domain architecture and probably promoter sequences; the high conservation among species indicated the ancient origin and diversification of the still evolving gene family. The comprehensive structural and expression characterization of the complete set of PDI and PDI-like wheat genes represents a basis for the functional characterization of this gene family in the hexaploid context of bread wheat. Description: L'articolo é disponibile sul sito dell'editore: http://www.biomedcentral.com Thu, 31 Dec 2009 23:00:00 GMT http://hdl.handle.net/2067/1609 2009-12-31T23:00:00Z http://hdl.handle.net/2067/1610 Title: Identification and validation of reference genes for quantitative RT-PCR normalization in wheat Authors: Paolacci, Anna Rita; Tanzarella, Oronzo A.; Porceddu, Enrico; Ciaffi, Mario Abstract: Background: Usually the reference genes used in gene expression analysis have been chosen for their known or suspected housekeeping roles, however the variation observed in most of them hinders their effective use. The assessed lack of validated reference genes emphasizes the importance of a systematic study for their identification. For selecting candidate reference genes we have developed a simple in silico method based on the data publicly available in the wheat databases Unigene and TIGR. Results: The expression stability of 32 genes was assessed by qRT-PCR using a set of cDNAs from 24 different plant samples, which included different tissues, developmental stages and temperature stresses. The selected sequences included 12 well-known HKGs representing different functional classes and 20 genes novel with reference to the normalization issue. The expression stability of the 32 candidate genes was tested by the computer programs geNorm and NormFinder using five different data-sets. Some discrepancies were detected in the ranking of the candidate reference genes, but there was substantial agreement between the groups of genes with the most and least stable expression. Three new identified reference genes appear more effective than the well-known and frequently used HKGs to normalize gene expression in wheat. Finally, the expression study of a gene encoding a PDI-like protein showed that its correct evaluation relies on the adoption of suitable normalization genes and can be negatively affected by the use of traditional HKGs with unstable expression, such as actin and α-tubulin. Conclusion: The present research represents the first wide screening aimed to the identification of reference genes and of the corresponding primer pairs specifically designed for gene expression studies in wheat, in particular for qRT-PCR analyses. Several of the new identified reference genes outperformed the traditional HKGs in terms of expression stability under all the tested conditions. The new reference genes will enable more accurate normalization and quantification of gene expression in wheat and will be helpful for designing primer pairs targeting orthologous genes in other plant species. Description: L'articolo è disponibile sul sito dell'editore: http://www.biomedcentral.com Wed, 31 Dec 2008 23:00:00 GMT http://hdl.handle.net/2067/1610 2008-12-31T23:00:00Z http://hdl.handle.net/2067/1617 Title: MIKC type genes of the MADS-box family in wheat: molecular and phylogenetic analysis. Authors: Paolacci, Anna Rita; Tanzarella, Oronzo A.; Porceddu, Enrico; Ciaffi, Mario Abstract: The MADS-box family of transcription factors play key roles in the control of development and signal transduction in eukaryotes (1). The MADS family contains a DNA-binding domain (MADS box) and includes two main lineages, type I and type II, both of which are present in plants, animals and fungi (2). Type II MADS-box proteins of plants,also named as MIKCtype proteins, possess three more domains than Type I MADS-box proteins: intervening (I) domain, keratinlike (K) domain and C-terminal (C) domain. In plants MIKC-type genes are involved in several important developmental processes, such as flower morphogenesis, ovule development, vegetative growth, embryogenesis and fruit formation, and through phylogenetic analysis based on sequence comparison, they have been classified into 13 subfamilies (3). The MIKC transcription factors controlling floral organ identity are the best characterized. Analysis of homeotic floral mutants of Arabidopsis resulted in the formulation of the ABCDE genetic model, which explains how the combined functions and organ-specific expression of five gene classes (A, B, C, D and E) specify the identity of the floral organs (sepals, petals, stamens and carpels), forming the four concentric whorls, and ovules (4,5). The most comprehensive cloning and characterization of MIKC genes of grasses have been carried out in maize and rice (6,7). In this study we extend a detailed characterization of the diversity and complexity of MIKC-type genes to hexaploid wheat. Mon, 31 Dec 2007 23:00:00 GMT http://hdl.handle.net/2067/1617 2007-12-31T23:00:00Z http://hdl.handle.net/2067/1612 Title: Molecular and phylogenetic analysis of MADS-box genes of MIKC type and chromosome location of SEP-lke genes in n wheat (Triticum aestivum L.). Authors: Paolacci, Anna Rita; Tanzarella, Oronzo A.; Porceddu, Enrico; Varotto, Serena; Ciaffi, Mario Abstract: Transcription factors encoded by MIKC-type MADS-box genes control many important functions in plants, including Xower development and morphogenesis. The cloning and characterization of 45 MIKC-type MADSbox full-length cDNA sequences of common wheat is reported in the present paper. Wheat EST databases were searched by known sequences of MIKC-type genes and primers were designed for cDNA cloning by RT-PCR. Full-length cDNAs were obtained by 5 and 3 RACE extension. Southern analysis showed that three copies of the MIKC sequences, corresponding to the three homoeologous genes, were present. This genome organization was further conWrmed by aneuploid analysis of six SEP-like genes, each showing three copies located in diVerent homoeologous chromosomes. Phylogenetic analysis included the wheat MIKC cDNAs into 11 of the 13 MIKC subclasses identiWed in plants and corresponding to most genes controlling the Xoral homeotic functions. The expression patterns of the cDNAs corresponding to diVerent homeotic classes was analysed in 18 wheat tissues and Xoral organs by RT-PCR, real time RT-PCR and northern hybridisation. Description: L'articolo è disponibile sul sito dell'editore: http://www.springerlink.com Sun, 31 Dec 2006 23:00:00 GMT http://hdl.handle.net/2067/1612 2006-12-31T23:00:00Z http://hdl.handle.net/2067/1611 Title: Phloem cytochemical modification and gene expression following the recovery of apple plants from apple proliferation Authors: Musetti, Rita; Paolacci, Anna Rita; Ciaffi, Mario; Tanzarella, Oronzo A.; Polizzotto, Rachele; Tubaro, Franco; Mizzau, Michela; Ermacora, Paolo; Badiani, Maurizio; Osler, Ruggero Abstract: Recovery of apple trees from apple proliferation was studied by combining ultrastructural, cytochemical, and gene expression analyses to possibly reveal changes linked to recovery-associated resistance. When compared with either healthy or visibly diseased plants, recovered apple trees showed abnormal callose and phloem-protein accumulation in their leaf phloem. Although cytochemical localization detected Ca2+ ions in the phloem of all the three plant groups, Ca2+ concentration was remarkably higher in the phloem cytosol of recovered trees. The expression patterns of five genes encoding callose synthase and of four genes encoding phloem proteins were analyzed by quantitative real-time reverse transcription- polymerase chain reaction. In comparison to both healthy and diseased plants, four of the above nine genes were remarkably upregulated in recovered trees. As in infected apple trees, phytoplasma disappear from the crown during winter, but persist in the roots, and it is suggested that callose synthesis/deposition and phloem-protein plugging of the sieve tubes would form physical barriers preventing the recolonization of the crown during the following spring. Since callose deposition and phloem-protein aggregation are both Ca2+-dependent processes, the present results suggest that an inward flux of Ca2+ across the phloem plasma membrane could act as a signal for activating defense reactions leading to recovery in phytoplasma-infected apple trees. Description: L'articolo é disponibile sul sito dell'editore: http://www.apsjournals.apsnet.org Thu, 31 Dec 2009 23:00:00 GMT http://hdl.handle.net/2067/1611 2009-12-31T23:00:00Z http://hdl.handle.net/2067/1621 Title: Protein disulfide isomerase gene family in wheat: genomic structure, phylogenetic and expression analyses. Authors: Ciaffi, Mario; Paolacci, Anna Rita; d'Aloisio, Elisa; Dhanapal, Arun Prabhu; Tanzarella, Oronzo A.; Porceddu, Enrico Abstract: PDI and PDI-like proteins are responsible for multiple metabolic functions, including secretory protein folding, chaperone activity and redox signalling. Most studies on their diversified metabolic roles have been carried out in mammalians, whereas in plants the knowledge on the structural and functional features of these proteins and of their encoding genes is much less extensive. The purpose of the present research was the cloning and characterization of the genes encoding PDI and PDI like proteins in bread wheat and the comparison of their structure and expression with those of homologous genes isolated in other plant species. Former studies in wheat and other cereal species had been restricted to the genes encoding the typical PDI, which may accomplish an important role in the folding and deposition of seed storage proteins. Since wheat flour quality is strongly affected by composition and structure of seed storage proteins, the potential involvement of the PDI and of some PDI-like proteins of wheat in the seed storage protein folding and in the formation of intra- and inter-molecular disulfide bonds makes their study particularly interesting. Fourteen wheat cDNA sequences of PDI-like genes were amplified and cloned; eight of them were relative to distinct PDI-like genes, whereas six corresponded to homoeologous sequences. Also the genomic sequences of the eight non-homoeologous genes were amplified and cloned. Phylogenetic analyses, which included the eight PDI-like genes cloned in this research and the typical PDI gene, assign at least one of them to each of the eight major clades identified in the phylogenetic tree of the PDI gene family of plants. Although not probable, the presence of additional wheat genes of the PDI family can not be ruled out. The genes of the wheat PDI family were located in chromosome regions syntenic with the chromosome locations of their rice homologs, confirming their close syntenic relationships. Within the same phylogenetic group a high level of conservation, in terms of sequence homology, genomic structure and domain organization, was detected between the wheat sequences and those of the compared plant species. The wheat proteins of five groups (I-V) have two thioredoxin-like active domains and show structural similarities to the corresponding proteins of higher eukaryotes, whereas those of the remaining three groups (VI-VIII) contain a single thioredoxin-like active domain. Phylogenetic analysis showed that the complete set of PDI and PDI-like genes was already present in P. patens and that extended phenomena of duplication events have characterized the evolution of this gene family in different plant taxa. The comparison of the exon/intron structure showed a very similar genomic organization across the analysed species, including P. patens, whereas the alga C. reinhardtii showed a different intron/exon structure. The high conservation level of sequence and genomic organization within the PDI gene family, even between distant plant species, might be ascribed to the key metabolic roles of their protein products. The expression analysis of the nine non-homoeologous wheat genes, which was carried out by quantitative real time RT-PCR (qRT-PCR) in a set of 29 samples including tissues, developmental stages and temperature stresses, showed their constitutive, even though highly variable transcription rate. The comprehensive structural and expression characterization of the complete set of PDI and PDI-like genes of wheat performed in this study represents a basis for future functional characterization of this gene family in the hexaploid context of bread wheat. Thu, 31 Dec 2009 23:00:00 GMT http://hdl.handle.net/2067/1621 2009-12-31T23:00:00Z http://hdl.handle.net/2067/1620 Title: ‘Recovery’ from apple proliferation disease: an integrated approach. Authors: Musetti, Rita; Paolacci, Anna Rita; Ciaffi, Mario; Tanzarella, Oronzo A.; Polizzotto, Rachele; Tubaro, Franco; Mizzau, Michela; Ermacora, Paolo; Badiani, Maurizio; Osler, Ruggero Abstract: Recovery is the spontaneous remission, sometimes permanent, from disease symptoms. Phytoplasmas surviving in the roots are not able to recolonise the plant crown. The causes that induce recovery remain still unknown and its physiological bases are poorly understood. In this research the modifications in the phloem tissue related to recovery-induced resistance in apple have been investigated through ultrastructural, chemical, cytochemical and gene expression analyses of leaf tissues from recovered, healthy and apple proliferation-diseased plants. Ultrastructural observations detected abnormal callose and P-protein accumulations in the phloem of recovered apple plants. Callose synthesis and Pprotein plugging, which are Ca2+-dependent, would form physical barriers preventing the in planta movement. The cytochemical localization by potassium pyroantimonate detected the presence of Ca2+ ions in the phloem in all the three groups of plants; however the Ca2+ concentration was remarkably higher in the cytosol of the recovered apple plants. This observation would support the hypothesis that resistance mechanisms would be related to an increased Ca2+- dependent signaling activities. Apple genes coding for callose synthases and phloem proteins were identified by an in silico approach. The expression patterns of five genes encoding callose synthases (MDCALS1/5) and of four genes encoding phloem proteins (MDPP2-1/3 and MDERG1) were analysed by quantitative real time RT-PCR. Four of the nine analysed genes were up-regulated in recovered plants in comparison to healthy and diseased ones, supporting the hypothesis that recovered apple plants were able to develop resistance mechanisms dependent from Ca2+ signal activities. Wed, 31 Dec 2008 23:00:00 GMT http://hdl.handle.net/2067/1620 2008-12-31T23:00:00Z