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 - 1.22

THE ISOLATION OF EARLY-COR GENES IN DURUM WHEAT REVEALS THE PRODUCTION OF ALTERNATIVELY SPLICED TRANSCRIPTS IN RESPONSE TO LOW TEMPERATURE

A.M. MASTRANGELO*, S. BELLONI**, S. BARILLI**, N. DI FONZO*, L CATTIVELLI**

*) Experimental Institute Cereal Research, Foggia (Italy)

**) Experimental Institute Cereal Research, Fiorenzuola d’Arda (Italy)

cold, drought, PCR-select, alternative splicing

The exposure of plant to abiotic stress determines a physiological and molecular reaction that depends, in initial phases, on a complex cascade of stress signal transduction in which few regulatory genes control many downstream genes that contribute to stress tolerance as a whole. Most of known cold-related genes in cereals belong to the group of downstream genes, beginning to be expressed after 20-24 hours of cold stress, and only few early induced genes with a regulatory role have been described. To elucidate the early molecular events in stress sensing and signal transduction, a suppression-subtractive library of control and cold stressed (6 hrs at 3°C) durum wheat samples has been constructed by using the Clontech PCR-select cDNA Subtraction Kit.

This work yielded twelve different clones reconstructed through RACE experiments. Three clones gave positive matches in database with genes already known to be involved in plant stress response (actin-binding protein gene, S-adenosylmethionine decarboxilase gene and a sequence coding for a farnesylated protein). Four clones showed similarity with genes never studied in relation to stress tolerance, a putative ribokinase, a putative ARM repeat containing protein, a putative transmembrane protein and a rice protein of unknown function. Three clones found matches only with bread wheat ESTs, and for the last two clones any significant similarity has been found in database. All these clones have been defined e-cor (early cold-regulated) genes, being over expressed or induced by cold within the first 6 hours of exposition to low temperature.

For some genes isolated in this work, the cold stress showed to influence not only, quantitatively, the abundance of transcripts but also the type of transcript present: a putative post transcriptional regulation mechanism based on alternative splicing has been found for two of isolated clones.

The gene coding for the putative ribokinase (7H8), characterised by a complex coding region containing 8 exons and 7 introns, revealed the production of different alternative transcripts, on the basis of the presence/absence of some introns, following the cold treatment. The majority of these transcripts appeared in a transient manner between four and eight hours of low temperature exposure. This mechanism has been found also in other cereals, bread wheat and barley, but not in homologous gene of the dicot Arabidopsis. The 6G2 clone, similar to a rice hypothetical protein, showed the production of two alternative transcripts, around four hours of cold treatment, containing or not a 100bp intron in the 3’ UTR region of the messenger.

The presence of the intron could influence some properties of the transcript like its stability or binding capacity to regulatory proteins or to the ribosome. Several examples are described in literature, in which multiple transcript forms are synthesised from a single pre-mRNA in response to biotic and abiotic stresses. In this cases the alternative splicing has a role in the regulation of stress tolerance, and it is not a simple target site for stress damage. So further studies are in progress in order to clarify the meaning of the production of 7H8 and 6G2 alternative transcripts as a putative mechanism for the post transcriptional regulation of gene expression in response to cold stress.