Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress

Verona, Italy - 24/27 September, 2003

ISBN 88-900622-4-X

Poster Abstract - 1.13

ARABIDOPSIS GENE EXPRESSION CHANGES DURING AND AFTER OZONE STRESS

N. TOSTI*, S. PASQUALINI**, E. FALISTOCCO**, G. DELLA TORRE**^,***, L. EDERLI**, F. PAOLOCCI*

*) Consiglio Nazionale delle Ricerche, Istituto di Genetica Vegetale, sezione di Perugia

**) Dipartimento di Biologia Vegetale e Biotecnologie Agroambientali, Università degli Studi di Perugia

***) Dipartimento di Arboricoltura e Protezione delle Piante, Università degli Studi di Perugia

GeneChip microarray, Arabidopsis, ozone

The concentration of tropospheric ozone (O₃) has increased during the past decades due to human activities, and it has been estimated that in year 2100, 50% of global forest will be exposed to potentially phytotoxic O₃ concentrations. Plants respond to high ozone levels showing both sensitivity, manifested as the appearance of rapid lesion formation, or tolerance. A. thaliana ecotype Col-0 is reported to be particularly tolerant to ozone and severe ozone fumigation did not induce any significant visual modification on rosette leaves and this makes this plant species of extreme interest to study genes involved into stress adaptive mechanisms. Therefore, to dissect the effects of ozone on plant gene expression profile, a microarray analysis was performed by means of GeneChip probe array developed by Affymetrix. The GeneChip contains more than 22,500 A. thaliana oligonucleotide probe sets, representing approximately 24,000 genes.

9-week A. thaliana old plants were exposed to a single dose of 300 ppb ozone for 6 h. Entire plant rosettes were harvested before (0h) and after 3 h (3h) of ozone treatment as well as after 6 h from the end of the treatment (12h). Plants after fumigation were left in growth chamber to recover.

Total leaf RNA was isolated from of 4 bulked plants for each of time points and used as probe for microarray. As control, time point 0h and 3h microarray analyses were carried out in duplicate using as probe newly isolated RNA.

The hybridising arrays, over a background level, ranged from 55-60 % for the control, to 62 % for 3h and 12h plants of the total probes. After 3 hours of treatment 12.28 % and 7.66 % of the total probes were up and down regulated, respectively. These percentages decreased down to 4.25 and 2.8 when only probes showing a two-fold difference with respect to the control were considered. The gene-related probes belonged to several gene families and many pathways resulted to be involved in response to ozone stress, while in some cases genes belonging to the same biosynthetic pathway were up and down regulated.

The log ratio of the expression values for each probe set between the control (0h) and treated samples (3h and 12h) was calculated. For each comparison, the probe sets that showed greater than two-fold difference from the control in at least one sample were selected. The expression values of the selected samples were subjected to a log₂ transformation then to a Self Organising Tree Algorhytm (SOTA). We found 29 gene clusters that were subdivided in four principal groups. An analysis of the SOTA dendrogram showed that in some cases genes belonging to the same gene family were clustered together. Moreover, the study of the promoter region of the up regulated genes showed the presence of over-represented motifs that were statistically related to some clusters of the SOTA dendrogram.