Introns are key regulatory elements of rice tubulin expression

The genomic clones containing elements that regulate transcription of the three known rice (Oryza sativa L.) alpha-tubulin isotypes (Ostua1, Ostua2 and Ostua3) have been isolated. We have used these genomic regions to identify the regulatory elements that contribute to the expression of a marker gene (gusA) in transient assays performed on rice calli derived from mature embryos. In all cases, we found that the first intron was required to achieve high levels of expression. This is consistent with data already reported for the alpha-tubulin isotype1 and indicates that a common regulatory mechanism is active on all the members of the rice alpha-tubulin gene family. The enhancing effect of the first intron was then tested by constructing illegitimate combinations of alpha-tubulin promoter and intron sequences (Ostua1pro-Ostua2intro; Ostua1pro-Ostua3intro; Ostua2pro-Ostua3intro; Ostua3pro-Ostua2intro) and then by assaying beta-glucuronidase (GUS) activity in transformed rice calli. All illegitimate combinations expressed GUS at high level, suggesting that rice alpha-tubulin promoters and introns can be exchanged among the different isotypes. This did not occur when the intron of the rice beta-tubulin isotype16, known to enhance transcription of its own gene, was used in place of the alpha-tubulin intron. We have also analysed the effect of abscisic acid (ABA) on GUS expression in rice calli transformed with chimeric tubalpha2pro-intro::gusA and tubalpha3pro-intro::gusA constructs. ABA was able to reduce GUS expression only in the presence of the tubalpha2pro-intro sequence. We discuss these data in terms of mechanisms that in rice, as opposed to other plants, may control tubulin isotype-specific expression and the involvement of ABA in the regulation of alpha-tubulin expression.