Phloem cytochemical modification and gene expression following the recovery of apple plants from apple proliferation disease.

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 Ca(2+) ions in the phloem of all the three plant groups, Ca(2+) 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 up-regulated 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 Ca(2+)-dependent processes, the present results suggest that an inward flux of Ca(2+) across the phloem plasma membrane could act as a signal for activating defense reactions leading to recovery in phytoplasma-infected apple trees.

Cloning and characterization of wheat PDI (protein disulfide isomerase) homoeologous genes and promoter sequences.

The genomic and cDNA sequences of three PDI homoeologous genes located on chromosomes 4A, 4B and 4D of bread wheat and their promoters were cloned and sequenced. The three sequences showed a very high conservation of the coding region and of the exon/intron structure, which consisted of ten exons. The comparison of wheat sequences with those of rice and Arabidopsis showed a significant conservation of the exon/intron structure across the three species. The expression of each gene was analysed by RT-PCR in different plant tissues (roots, coleoptiles, spikelets, leaves and developing caryopses). All the genes showed a higher expression in developing caryopses than in other analysed tissues, wherein some differences were detected. The promoter sequences of the three genes possessed some regulatory motifs typical of endosperm specific expression.

Isolation and characterization of S genome specific sequences from Aegilops sect. sitopsis species.

Three S genome specific sequences were isolated from Aegilops sect. sitopsis species using different experimental approaches. Two clones, UTV86 and UTV39, were isolated from a partial genomic library obtained from DNA of Aegilops sharonensis, whereas a third clone, UTV5, was isolated from Aegilops speltoides. The three clones were characterized by sequencing, analysis of methylation, and sequence organization and abundance in some Aegilops and Triticum species. The clones UTV39 and UTV5 belong to the same family of tandem repeated sequences and showed high homology with a sequence already present in nucleotide databases. The UTV86 clone from Ae. sharonensis corresponded to an interspersed low frequency repeated sequence and did not show any significant homology with reported sequences. Southern hybridization experiments, using the cloned sequences as probes, detected polymorphism in the restriction patterns of all the five Aegilops species in section sitopsis. Aegilops speltoides showed the most divergent hybridization pattern. A close relationship was detected between the S genome of Ae. speltoides and the G genome of the wild Triticum timopheevii. In situ hybridization revealed a telomeric and (or) subtelomeric location of the sequences UTV39 and UTV5.

Molecular characterization of gene sequences coding for protein disulfide isomerase (PDI) in durum wheat (Triticum turgidum ssp. durum).

The organisation of the durum wheat genomic sequence (3.5 kb) coding for protein disulfide isomerase (PDI), deduced by comparison between genomic fragments and cDNA sequences (1.5 kb) isolated from immature caryopses, is described. The gene structure consists of ten exons and nine introns. The presence of consensus sequences involved in splicing, such as intron-exon junctions and branchpoint, has been observed and discussed. Although the deduced wheat PDI amino acid sequence exhibited an overall identity of only 31% to that of human PDI, their modular architecture in terms of number, size, location and secondary structure-propensities of the constituent domains are remarkably similar. The comparison of the amino acid sequences with the eight available plant PDI-like sequences showed a high identity with four of them and low with the remaining ones. Analyses of transcription levels showed that the PDI mRNA was present in all analysed tissues, with much higher expression in immature caryopses.

Rapid and efficient detection of genetic polymorphism in wheat through amplification by polymerase chain reaction.

The polymerase chain reaction (PCR) was used to amplify genomic DNA of several wheat genotypes. The oligonucleotides used as primers were the terminal sequences of a gamma-gliadin gene. The electrophoretic analysis of the PCR products showed specific bands which revealed both inter- and intra-specific genetic polymorphism among the examined genotypes. The technique is proposed as a very simple and efficient alternative to RFLP markers.

Morphology and chromosome pairing of a hybrid between Triticum durum Desf. and Haynaldia villosa (L.) Schur.

Intergeneric hybrids between T. durum Desf. (2n = 4x = 28, AABB) and Haynaldia villosa (L.) Schur. (2n = 2x = 14, VV) was obtained at a frequency of about 5.6% of pollinated florets. Phenotypically the f1 plants resemble more the maternal parent than the H. villosa and are almost completely sterile. However, some seeds were obtained on selfed and backcrossed heads with the durum wheat parent. The hybrid had a somatic complement of 2n = 3x = 21, ABV, with a mean chromosomal relationship of 13.62 univalents, 3.30 bivalents, and 0.26 trivalents. The high pairing was likely due to gene(s) of H. villosa interacting with the 5B homoeologous restricting system of wheat.