CONICET | Buscador de Institutos y Recursos Humanos

The promoters of the Arabidopsis thaliana nuclear genes COX5b-1 (At3g15640) and COX5b-2 (At1g80230), encoding subunit 5b of mitochondrial cytochrome c oxidase, were analyzed using plants transformed with different promoters fragments fused to a reporter gene GUS. Quantitative measurements in extracts prepared from 21-day-old plants carrying the entire promoters regions showed that the inclusion of sucrose, the ethylene precursor ACC, phosphate and H2O2 in the culture medium induced both COX5b-1 and COX5b-2 promoter-dependent b-glucuronidase expression. In addition, the inclusion of 6-BAP (cytokinin), abscisic acid (ABA) and gibberellic acid (GA) increase only COX5b-1:GUS expression while the auxin indole-3-acetic acid (IAA) or irradiation with UV-B light enhance COX5b-2:GUS expression levels. On the other hand, measurements of relative transcript levels in wild-type non-transformed plants using real-time PCR suggesting that these compounds are effectors of the endogenous genes. The analysis of progressive upstream deletions of the promoters led us to identify fragments required for these responses: in COX5b-1 nucleotides -2000 to -600 from the translation start site for GA and phosphate responses, 600/400 for ethylene response, -400/-350 for cytokinin response and -350/-260 for sucrose and ABA responses. Site-directed mutagenesis showed that over-represented TCAT elements between nucleotides -330 and -290 are required for sucrose response and two CCACTTG elements that resemble the distal portion of the B-box, present at -281 and -263, are required for ABA-enhanced expression. In COX5b-2 nucleotides -1000/-650 are required for ethylene and IAA responses and -650/-400 for phosphate and H2O2 responses. Site-directed mutagenesis showed that two site II elements, present at -172 and -148, are essential for sucrose response while a G-box/ABRE (GACACGTG) present at -660 is necessary for UV-B response

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