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

Salsomaggiore Terme, Italy - 26/29 September, 2001

ISBN 88-900622-1-5

Poster Abstract

IDENTIFICATION OF A NEW CIS-ELEMENT RESPONSIVE TO LOW TEMPERATURE IN BARLEY

CROSATTI C., GOVONI C., MAZUCCOTELLI E., GROSSI M.

Istituto Sperimentale per la Cerealicoltura, Sezione di Fiorenzuola, Via S. Protaso 302, I-29017 Fiorenzuola d’Arda (PC), Italy

c.crosatti@iol.it

barley, promoter analysis, cold acclimation

To understand the regulation mechanisms leading to the cold response we have analysed the putative promoter region (650 bp upstream of the initiation codon) which control the expression of the barley cold regulated gene cor14b. This gene is induced in leaf cells exclusively during low temperature treatment; cor14b encodes a protein localised into the stroma fraction of the chloroplast. In a preliminary experiment the whole putative promoter sequence fused with the uidA gene as reporter, was assayed by transient expression after biolistic particle bombardment into barley leaves The activity of the promoter, revealed via b-glucoronidase assay, was displayed in leaves exposed at low temperature and no activity was detected at 20°C. Furthermore the cor14b promoter- uidA construct was not active when transiently expressed in leaves of albino plants and in roots, two features that resemble the expression behaviour of the cor14b gene. These results demonstrated that the 650bp promoter fragment under investigation contains all cis-elements necessarily to drive the tissue and low temperature–dependent expression of cor14b.

A comparative analysis of known cis-responsive element with the sequence of cor14b promoter revealed the presence of several regions similar, although not identical to the cold responsive element found in Arabidopsis, Brassica and Hordeum. To prove the activity of these motifs seven chimeric cor14b-promoter/uidA constructs were produced in which the putative cis-elements were deleted one by one. A 27bp deletion (from position -274 to position –247) lead to a major decrease in the cold-dependent activity of the promoter indicating the presence active cis-elements. The 27bp sequence contains a six bp motif (CCCAAA) similar to a previously described Low Temperature Responsive Element of the barley gene blt4.9 (CCGAAA). New constructs carrying base substitutions in the 27bp region were therefore produced. Transient expression analyses of the base substitution constructs showed that mutations at the CCCAAA motif did not modify the promoter activity. On the contrary, mutations in a flanking sequence (GGTACG) strongly impaired the cold induce activity of reported gene suggesting that GGTACG may represent a novel cold responsive cis-elements.