Isolamento e caratterizzazione di sequenze cDNA indotte da ozono in frumento duro
Author(s)
Acampora, Andrea
Date Issued
August 27, 2008
Type
Doctoral Thesis
Abstract
In the cultivation area of durum wheat in Italy, there are high levels of photochemical pollution by ozone, in particular during heading time and seed development. The apparent resistance of wheat to ozone is due to light foliar symptoms, but the effects on grain productivity are relevant, due to early foliar senescence during the most sensitive period of grain filling. The damage extent is obviously related to pollutant concentrations and atmospheric characteristics, but there is also a significant intraspecific variation for ozone tolerance. Molecular aspects of the response to ozone stress are well known in Arabidopsis, whereas very little is known in monocots and wheat. Durum wheat production is economically relevant in Italy and the possible damages produced by the photochemical pollution justify research programmes on the biochemical and molecular mechanisms involved in the response of this species to ozone exposure.
In particular the research project included the following three phases: 1) evaluation of 16 durum wheat cultivars for tolerance/susceptibility to O3 by exposure in controlled environment; 2) identification of cDNA-AFLPs induced by O3, using one of the tolerant durum wheat cultivars; 3) sequencing of the induced sequences and expression analysis by RT-PCR in tolerant and susceptible genotypes.
Seedlings were exposed to acute ozone treatment under controlled conditions. Evaluation of the appearance of the necrotic lesions was carried out at the end of the O3 exposure and after 5, 24 and 48 h and their incidence was evaluated by the MacKinney Index. On the basis of leaf damage and of some physiological parameters (gas exchange measurements, leaf chlorophyll fluorescence, membrane solute leakage and relative water content), the O3 tolerant cv Portorico and, during the first stage of activity, the O3-sensitive cv Duilio, have been selected for differential analysis by cDNA-AFLP. For some of the isolated sequences expression analyses have been carried out in six varieties of durum wheat, showing contrasting responsiveness to O3. The expression profiles of the transcripts from two-week old ozonated and control plants were compared by a modified cDNA-AFLP technique. The analysis of about 2500 amplified transcripts from ozonated and control plants detected 165 differential products induced by O3. The nucleotide and deduced amino acid sequences of 122 out of 165 O3-induced clones, whose differential or preferential expression in ozonated plants had been verified by RT-PCR analyses, were searched in the online databases of DNA and protein sequences. Significant homology was found for 88 differential products: 9 detected homologies only within the wheat ESTs, indicating, most likely, their high specificity for this species, whereas 79 found homologous sequences in all the searched databases. No significant homology was detected for the remaining 34 clones. On the basis of their homology with sequences in the databases with known functions, the 79 clones were assigned to seven functional groups: I) signal transduction (15 clones); II) cell organization (9); III) protein synthesis/destination/degradation (9); IV) metabolism (11); V) cell rescue/defence (12); VI) transcription (7); VIII) unknown (16). It is noteworthy that only 13 clones corresponded to wheat sequences reported in DNA and protein databases, and 44 were found within the EST sequences of wheat. Forty-five of the 63 isolated AFLP-tags, found to match genes with known functions, had been previously recognized as O3-responsive in Arabidopsis. On the basis of these results it can be inferred that the modified cDNA-AFLP procedure adopted in this research was effective in detecting O3-responsive genes. The lack in the Arabidopsis transcriptome of some sequences cloned from durum wheat might be explained either by differences between taxonomically distant species or by the different conditions of ozone exposure adopted.
The expression analysis of the 63 sequences induced by ozone which showed significant homology with genes with known function were carried out by RT-PCR in the six durum wheat varieties selected for their contrasting response to ozone stress. On the basis of these analyses the 63 sequences have been classified in four groups: the first group includes 25 sequences that showed similar expression patterns in all the six tolerant and susceptible cvs after exposure to O3; the second one comprises 12 sequences preferentially induced or overexpressed only in tolerant cvs; the third is composed of 6 sequences that were preferentially induced or overexpressed only in susceptible cvs; the forth group includes the remaining 20 sequences that showed very complex expression patterns in the six analysed genotypes. It is noteworthy that the expression patterns of some sequences are directly related with the contrasting responsiveness to O3 of the six varieties. These sequences could therefore represent an important starting point for studies of the molecular mechanisms of tolerance/susceptibility to ozone in durum wheat.
Additional information
Dottorato di ricerca in Genetica agraria
File(s)![Thumbnail Image]()
Name
aacampora_tesid.pdf
Size
5.07 MB
Format
Adobe PDF
Checksum (MD5)
41ed7c4c9c7ae25455236a34cf919844