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Different rice alpha-tubulin isotypes share a common mechanism for transcriptional regulation

 
: Fiume, E.; Christou, P.; Breviario, D.

Lupotto, E. ; Fondazione per le Biotecnologie:
ricEUconf. Dissemination conference of current European research on rice 2002. Book of abstracts : June 6-8, 2002
Torino, 2002
pp.55-56
Dissemination Conference of Current European Research on Rice <2002, Torino>
English
Conference Paper
Fraunhofer IME ()
cytoskeleton; promoter; tubulin; gene regulation

Abstract
Microtubules are proteinaceous filaments composed of heterodimers of alpha and beta tubulins and MAPs (Microtubule-associated proteins). Microtubules play a fundamental role in many aspects of eukaryotic cell biology, including cell division, cytoplasmic organisation, vesicular transport, response to biotic and abiotic stresses, signal transduction and cell morphology (Nogales, 2000). In plant, cell elongation and polarity of growth depend on plant-specific microtubular arrays. Plants contain more expressed tubulin genes than animals. These genes are grouped in two small families: alpha and beta, each of them comprising different isotypes. Specific sequence features further divide the alpha tubulin gene family into subfamilies: I and II. In rice evidence has been found for 3 alpha tubulin isotypes and 6-7 beta tubulin isotypes. In a number of plant species it has been widely demonstrated that different isotypes of alpha and beta tubulin exhibit different expression patterns that are regulated spatially and temporally. Some tubulin isotypes are characterised by a near constitutive pattern of expression, whereas others may be expressed in specific growth phases, tissues or in response to specific stimuli. These patterns of differential expression can be substantially associated to different promoters acting upstream of the coding regions. We are interested in understanding the molecular basis of tubulin-isotype differential gene regulation. We have screened a rice genomic library and have isolated three clones containing the genomic sequences of all the alpha tubulin genes so far known to be present in rice. We isolated the putative regulatory regions of these alpha tubulin genes (alpha1, alpha2 and alpha3) and cloned them into a promoter-less pBI221 backbone vector upstream of the gus A reporter gene. Through transient expression experiments performed on mature rice embryos (Oryza sativa CV Bengal) we discovered that each alpha tubulin gene requires the presence of the first intron of the coding sequence for maximum expression. GUS activity was shown to be approximately 50 per cent of what is normally obtained when the maize ubiquitin 1 plus its first intron are used to drive expression. Deletion of the first intron of the coding sequence gave rise to a dramatic decrease in the level of GUS expression (less than 30 per cent of its original activity). This was verified for all the rice alpha tubulin isotypes. Such an enhancing effect seems to be independent of the specificity of each alpha tubulin isotype promoter sequence. In fact, recombination of different alpha tubulin promoters with their own or other alpha tubulin first introns consistently yielded high and comparable levels of expression. We also combined the alpha3 promoter region (upstream from its own ATG) with the rice beta16 tubulin intron, that is located in the leader sequence of the beta16 isotype and it was demonstrated to increase of 5-10 fold over the activity of its native promoter (Morello et al. 2002). In contrast of what we reported above for alpha tubulin promoter-intron combinations, the combination of an alpha tubulin promoter with a beta16 regulatory intron did not produce any appreciable effect on GUS expression. The promoter activity of this alpha-beta hybrid construct was even lower than the activity exhibited by constructs carrying only the alpha3 promoter. This result suggests that alpha and beta tubulin gene expression may depend on different regulatory mechanisms even though both rely on introns to achieve high level of expression. Previous findings in transgenic rice plants demonstrated that the first intron of the rice alpha1 tubulin sustained high levels of GUS activity in meristematic tissues. Constructs lacking the tubulin intron showed constitutive but low level of expression (Jeon et al., 2000). A number of alpha tubulin promoter regions have been isolated from several plant species (e.g. maize, barley, Arabidopsis). These promoters conferred a tissue specific pattern in transgenic plants. However use of the alpha tubulin introns was not investigated. Our results suggest that alpha tubulin promoter-intron combinations are important in terms of understanding in depth regulatory elements that control levels ant tissue specificity of genes driven by these elements.

: http://publica.fraunhofer.de/documents/B-73332.html