Silvia Gianì

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 α-tubulin isotype1 and indicates that a common regulatory mechanism is active on all the members of the rice α-tubulin gene family. The enhancing effect of the first intron was then tested by constructing illegitimate combinations of α-tubulin promoter and intron sequences (Ostua1pro–Ostua2intro; Ostua1pro–Ostua3intro; Ostua2pro–Ostua3intro; Ostua3pro–Ostua2intro) and then by assaying β-glucuronidase (GUS) activity in transformed rice calli. All illegitimate combinations expressed GUS at high level, suggesting that rice α-tubulin promoters and introns can be exchanged among the different isotypes. This did not occur when the intron of the rice β-tubulin isotype16, known to enhance transcription of its own gene, was used in place of the α-tubulin intron. We have also analysed the effect of abscisic acid (ABA) on GUS expression in rice calli transformed with chimeric tubα2pro-intro::gusA and tubα3pro-intro::gusA constructs. ABA was able to reduce GUS expression only in the presence of the tubα2pro-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 α-tubulin expression.

Molecular cloning of two novel rice cDNA sequences encoding putative calcium-dependent protein kinases

We have isolated, from a cDNA library constructed from rice coleoptiles, two sequences, OSCPK2 and OSCPK11, that encode for putative calcium-dependent protein kinase (CDPK) proteins. OSCPK2 and OSCPK11 cDNAs are related to SPK, another gene encoding a rice CDPK that is specifically expressed in developing seeds [20]. OSCPK2 and OSCPK11-predicted protein sequences are 533 and 542 amino acids (aa) long with a corresponding molecular mass of 59436 and 61079 Da respectively. Within their polypeptide chain, they all contain those conserved features that define a plant CDPK; kinase catalytic sequences are linked to a calmodulin-like regulatory domain through a junction region. The calmodulin-like regulatory domain of the predicted OSCPK2 protein contains 4 EF-hand calcium-binding sites while OSCPK11 has conserved just one canonical EF-hand motif. In addition, OSCPK2-and OSCPK11-predicted proteins contain, at their N-terminal region preceding the catalytic domain, a stretch of 80 or 74 residues highly rich in hydrophilic amino acids. Comparison of the NH2-terminal sequence of all three rice CDPKs so far identified (OSCPK2, OSCPK11 and SPK) indicates the presence of a conserved MGxxC(S/Q)xxT motif that may define a consensus signal for N-myristoylation. OSCPK2 and OSCPK11 proteins are both encoded by a single-copy gene and their polyadenylated transcripts are 2.4 and 3.5 kb long respectively. OSCPK2 and OSCPK11 mRNAs are equally abundant in rice roots and coleoptiles. A 12 h white light treatment of the coleoptiles reduces the amount of OSCPK2 mRNA with only a slight effect on the level of OSCPK11 transcript. With anoxic treatments, OSCPK2 mRNA level declined significantly and promptly while the amount of OSCPK11 transcript remained constant.

High polymorphism and resolution in targeted fingerprinting with combined β-tubulin introns

Tubulin-Based-Polymorphism (TBP) was originally introduced as a novel method for assaying genetic diversity in plants. TBP is based on polymorphism resulting from the PCR-mediated amplification of the first intron in the coding region of the β-tubulin gene family. Although, the method was successful in genetic assessment of some plant species and varieties, it suffered from low number of molecular markers due to limited variation in the first intron of β-tubulin gene family. We have now rectified this limitation by introducing the second intron of the β-tubulin genes as a valuable source of molecular markers. We show that the combined use of the two introns substantially increases the number of molecular markers and results in a reliable assessment of species/varieties relationships. After a preliminary validation on Brassica, this new combinatorial method was tested on species of Eleusine and Arachis. For both, reliable assessment of species relationships were obtained that were consistent with recently published studies resulting from more elaborated methods including DNA sequencing. Combinatorial TBP is a reliable, reproducible, simple, fast, and easy to score method that is very useful for breeding programs and species and variety assessments.

Overexpression of the calcium-dependent protein kinase OsCDPK2 in transgenic rice is repressed by light in leaves and disrupts seed development

Independent transgenic rice lines overexpressing the rice CDPK isoform OsCDPK2 were generated by particle bombardment. High levels of OsCDPK2 were detected in leaves removed from etiolated plants, as well as in stems and flowers. However, there was no overexpression in green leaves that had been exposed to light, confirming that OsCDPK2 protein stability was subject to light regulation. The morphological phenotype of transgenic plants producing high levels of recombinant OsCDPK2 was normal until the onset of seed development. Flowers developed normally, producing well-shaped ovaries and stigmas, and mature anthers filled with pollen grains. However, seed formation in these plants was strongly inhibited, with only 3–7% of the flowers producing seeds. Seed development was arrested at an early stage. We discuss these data with respect to the possible requirement for specific CDPK isoforms during rice seed 4.4ptdevelopment.

In rice, Oryzalin and abscisic acid differentially affect tubulin mRNA and protein levels

Abstract. The effect of the anti-microtubular drug Oryzalin (3,5-dinitro-N4,N4-dipropylsulfanilamide) on growth and elongation of rice (Oryza sativa L. cv. Arborio) roots and coleoptiles was investigated. At 100 nM, Oryzalin strongly reduced primary root elongation, caused loss of cell anisotropy and the disappearance of the cortical microtubule array. Under these conditions the amounts of α- and β-tubulin protein, but not mRNA, were heavily reduced. Similar data were also obtained in coleoptile segments treated with different concentrations of Oryzalin. However, when coleoptile elongation was inhibited by cis-abscisic acid, remarkable decreases in α- and β-tubulin accumulation were observed to occur at the mRNA level but not at the protein level. The transcriptional decreases could be reversed by re-addition of 3-indole acetic acid. Altogether, these data indicate that rice tubulin accumulation can be controlled at different levels, mRNA or protein, in response to Oryzalin or abscisic acid treatments.

Testing the IMEter on rice introns and other aspects of intron-mediated enhancement of gene expression

In many eukaryotes, spliceosomal introns are able to influence the level and site of gene expression. The mechanism of this Intron Mediated Enhancement (IME) has not yet been elucidated, but regulation of gene expression is likely to occur at several steps during and after transcription. Different introns have different intrinsic enhancing properties, but the determinants of these differences remain unknown. Recently, an algorithm called IMEter, which is able to predict the IME potential of introns without direct testing, has been proposed. A computer program was developed for Arabidopsis thaliana and rice (Oryza sativa L.), but was only tested experimentally in Arabidopsis by measuring the enhancement effect on GUS expression of different introns inserted within otherwise identical plasmids. To test the IMEter potential in rice, a vector bearing the upstream regulatory sequence of a rice β-tubulin gene (OsTub6) fused to the GUS reporter gene was used. The enhancing intron interrupting the OsTub6 5′-UTR was precisely replaced by seven other introns carrying different features. GUS expression level in transiently transformed rice calli does not significantly correlate with the calculated IMEter score. It was also found that enhanced GUS expression was mainly due to a strong increase in the mRNA steady-state level and that mutations at the splice recognition sites almost completely abolished the enhancing effect. Splicing also appeared to be required for IME in Arabidopsis cell cultures, where failure of the OsTub6 5′ region to drive high level gene expression could be rescued by replacing the poorly spliced rice intron with one from Arabidopsis.

Rice β-tubulins mRNA levels are modulated during flower development and in response to external stimuli

Abstract Rice β-tubulin cDNA clones have been recently isolated by different laboratories (Kang et al. (1994) Plant Mol. Biol. 26, 1975–1979; Breviario et al. (1995) Plant Physiol. 108, 823–824; Koga-Ban et al. (1995) DNA Res. 2, 21–26). Analysis of their deduced amino acid sequences shows the conservation of all those structural motifs typical of plant β-tubulins. Putative sequences for autoregulation and tubulin mRNA stability, for GTP binding and exchange as well as for herbicides and Ca 2+ binding are present. With the use of a generic or isotype specific β-tubulin (OS-TUB16) probe we assayed the level of transcriptional accumulation in different rice tissues, during flower development, cell elongation and in response to anoxia. We found enhanced levels of total or isotype-16 specific β-tubulin mRNA at early stages of flower development in spikelets at 8–10 days before anthesis. The lowest levels were observed in adult leaves. Total β-tubulin as well as isotype-16 specific transcript levels were strongly decreased (10-fold) in rice coleoptiles treated with 50 μM abscisic acid (ABA); a more modest decrease (2-fold) in the total level of rice α-tubulin mRNA was instead observed in these samples. We also show a strict correlation between growth under anoxia and β-tubulin transcriptional abundance. Rice roots, unable to grow in anoxia, showed very low amount of total β-tubulin mRNA whereas rice coleoptiles, capable of elongating under anaerobiosis, maintained high levels of total β-tubulin transcripts. However, with the use of a 3′ non-coding probe specific for β-tubulin isotype-16, we found that the amount of this transcript is always decreased by anoxia regardless the type of tissue analyzed.

Multiple tubulins: evolutionary aspects and biological implications

Plant tubulin is a dimeric protein that contributes to formation of microtubules, major intracellular structures that are involved in the control of fundamental processes such as cell division, polarity of growth, cell-wall deposition, intracellular trafficking and communications. Because it is a structural protein whose function is confined to the role of microtubule formation, tubulin may be perceived as an uninteresting gene product, but such a perception is incorrect. In fact, tubulin represents a key molecule for studying fundamental biological issues such as (i) microtubule evolution (also with reference to prokaryotic precursors and the formation of cytomotive filaments), (ii) protein structure with reference to the various biochemical features of members of the FstZ/tubulin superfamily, (iii) isoform variations contributed by the existence of multi-gene families and various kinds of post-translational modifications, (iv) anti-mitotic drug interactions and mode of action, (v) plant and cell symmetry, as determined using a series of tubulin mutants, (vi) multiple and sophisticated mechanisms of gene regulation, and (vii) intron molecular evolution. In this review, we present and discuss many of these issues, and offer an updated interpretation of the multi-tubulin hypothesis.

Plant tubulin intronics

Introns of plant tubulin genes are useful molecular tools to study IME (Intron Mediated Enhancement of gene expression) and to define plant genetic and evolutionary relationships through ILP (Intron Length Polymorphism). Here we show that the intron present within the 5′UTR sequence of some rice β‐tubulin genes can sustain IME in rice transgenic plants and that degenerated oligonucleotide mixtures designed to amplify the first and the second intron present within the coding sequence of plant β‐tubulin genes can successfully detect ILPs among different bean varieties.

Tubulin intron sequences: multi-functional tools

Control of plant and -tubulin gene expression is a key process involved in modulating the amount of tubulin in response to changes occurring in microtubule organization and dynamics. Accordingly, external signals or drugs that may interfere with microtubule assembly can determine changes in the level of tubulin expression. Regulation can occur at both transcriptional and translational level and can sometime involve peculiar mechanisms of control (Anthony and Hussey 1998; Breviario and Nick, 2000; Ebel et al., 2001). Recently, it was demonstrated that microtubule depolymerization caused by oryzalin, an antimitotic drug, can lead to a strong decrease in the amount of both and -tubulin. This regulation is mainly exerted at the protein level and involves a reduced rate of protein neo-synthesis as well as increased degradation of the unassembled tubulin polypeptides (Giani et al., 2002). On the other hand, studies performed with genomic region of the rice -tubulin isotype 1 have shown that the first intron present within the coding region is important for providing a high level of expression of a GUS reporter gene in meristematic tissues of transgenic rice plants (Jeon et al., 2002). Constructs lacking this intron showed constitutive, but low level of GUS gene expression. Preliminary data obtained on two additional rice -tubulin isotypes (tuba2 and tuba3) seem to confirm those findings (unpublished results). A similar situation can also be observed while analysing the regulatory sequence upstream from the ATG codon that controls the expression of -tubulin isotype 16 (Fig. 1). As shown for -tubulins, the presence of a long intron within the genomic sequence corresponding to the 5#UTR of the gene is capable of increasing the expression of the GUS reporter gene driven by pro 16 promoter in transient assays performed on rice calli derived from the mature * Corresponding author. Tel.: +39-022-369-9441; fax: +39-022-369-9411. E-mail address: diego@ibv.mi.cnr.it (D. Breviario).