Diego Breviario

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.

Functional analysis of DNA sequences controlling the expression of the rice OsCDPK2 gene

Plant calcium-dependent protein kinases (CDPKs) are involved in calcium-mediated signal transduction pathways. Their expression is finely tuned in different tissues and in response to specific signals, but the mechanism of such a regulation is still largely unknown. OsCDPK2 gene expression is modulated in vivo during rice (Oryza sativa L.) flower development and is downregulated by white light in leaves. In order to identify OsCDPK2 regulatory sequences, we amplified and cloned both the 5′ and 3′-flanking regions of the gene. Sequence analysis revealed that the leader sequence is interrupted by an intron, whose regulatory role was investigated. Different ß-gucuronidase (GUS) expression vectors, carrying combinations of the putative OsCDPK2 regulatory regions, were generated and GUS expression was analyzed both in transient assays and in transgenic rice plants. The whole 5′-flanking sequence was able to drive GUS expression in rice calli and leaves transiently transformed with the biolistic technique. Analysis of the GUS expression pattern in transgenic plants revealed strong activity in root tips, leaf veins and mesophyll cells, in flower reproductive organs and in mature pollen grains. Expression was also shown to be subject to an intron-mediated enhancement (IME) mechanism, since the deletion of the leader intron sequence from chimeric OsCDPK2::GUS plasmids almost completely abolished GUS activity. Furthermore, in transiently transformed leaves, GUS expression driven by the OsCDPK2 promoter-leader region was constitutively observed regardless of light or dark exposure. Light-regulated expression was restored by inserting the OsCDPK2 3′ untranslated region (3′UTR) downstream of the chimeric OsCDPK2::GUS transcription unit, suggesting that light down-regulation is mediated by a mechanism driven by the 3′UTR.

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.

h-TBP: an approach based on intron-length polymorphism for the rapid isolation and characterization of the multiple members of the β-tubulin gene family in Camelina sativa (L.) Crantz

We have developed a new version of the cTBP (combinatorial tubulin-based polymorphism) method, a previously described approach based on intron-length polymorphism (ILP), to rapidly characterize the β-tubulin gene family of Camelina sativa (L.) Crantz, a plant species of importance for oil production but still largely unexplored at genomic level. The method, named h-TBP, allows the rapid cloning of the β-tubulin genomic sequences that encompass the two introns, invariantly present at fixed positions within the coding region of the vast majority of the plant species. The β-tubulin sequences cloned by h-TBP also comprise part of exon1 and exon3 and the whole sequence of exon2. The h-TBP method has then been used to isolate, clone and characterize the β-tubulin gene family of C. sativa, composed of at least 20 different β-tubulin isotypes, named CsTUB1 through CsTUB20. The relatively high number of β-tubulin genes has been further substantiated by Southern-blot analysis. Comparison of the β-tubulin exon sequences of C. sativa with those of Arabidopsis thaliana, the closest relative among crucifers, defines distinct groups of putative orthologous genes, identified by a UPMGA cluster analysis. Analysis of the C. sativa β-tubulin intron sequences reveals some molecular features that can provide the first hints for the understanding of intron plasticity and evolution. From a more immediate perspective, these data provide the first substantial contribution to the characterization of the largely unexplored genome of C. sativa, and the tools for assisting programmes of breeding and selection of the most productive plants.