Laura Morello

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.

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.

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.

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.

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).

Effect of the different dimeric forms of the platelet‐derived growth factor on cellular responses in mouse Swiss 3T3 fibroblasts

PDGF consists of two polypeptide chains, A and B, and all three possible dimers have been isolated from different sources. Human PDGF, essentially AB, porcine PDGF (BB) and recombinant PDGF‐AA were tested on Swiss 3T3 fibroblasts for their ability to stimulate mitogenesis, phosphoinositide turnover and tyrosine phosphorylation of the PDGF receptor. When used in saturating amounts, the three isoforms were equally active in inducing mitogenesis. However, PDGF‐AA was less active than AB and BB to induce the phosphorylation of the receptor and the turnover of phosphoinositides (30% and 50%, respectively). These findings suggest that, in Swiss 3T3 fibroblasts, PDGF receptors of the α‐type are present in a slightly lower amount than β‐type. In addition, the two types of receptor appear to have similar physiological functions.

Dissociation of the ligand and dephosphorylation of the platelet-derived growth factor receptor

The ligand‐induced phosphorylation of the platelet‐derived growth factor (PDGF) receptor was followed at 37°C by a rapid dephosphorylation which was roughly parallel to the down regulation of the 125I‐PDGF binding sites. At 4°C, when the ligand‐receptor complexes remain associated with the cell surface, the phosphorylated form of the receptor was more stable. However if the ligand was dissociated from the receptor by means of a mild acid wash or a treatment with suramin, the dephosphorylation of the receptor also occurred at a low temperature. These data suggest that, due to the dissociation of the ligand, the kinase activity of the receptor is switched off so that the phosphotyrosine‐containing receptors remain exposed to the action of phosphatases that rapidly dephosphorylate them.

Traceback identification of plant components in commercial compound feed through an oligonucleotide microarray based on tubulin intron polymorphism

According to EU Regulations, all components of commercial compound feed need to be declared on the label. Effective protection against fraud requires severe controls based on accurate analytical methods to ascertain what is declared by the producers. The aim of this work was to develop an oligonucleotide microarray for the molecular recognition of multiple plant components in commercial feeds. We tested the potential of the highly polymorphic first intron sequences from members of the plant β-tubulin gene family as a target for plant DNA identification. 23 oligonucleotide capture probes, targeting species-specific intron sequences, were assembled within a low density microarray for the identification of 10 plant species, selected from among those most commonly used in cattle feed formulation. The ability of the array to detect specific components in complex flour blends and in compound feed was evaluated.

The Influence of Anaerobiosis on Membrane-Associated Rice (O. sativa L.) Protein Kinase activities

Summary Root and coleoptile membrane extracts of rice seedlings that were incubated aerobically or shifted to anoxia for 2 hours were compared for their endogenous protein kinase activities with the use of in vitro assays. The anoxic extracts showed a decreased labelling of a polypeptide of 73 kDa. This reduction was more evident in roots than in coleoptiles. The pattern of in vitro phosphorylated proteins obtained from membranes of rice coleoptiles cut from 8-day-old seedlings directly germinated in anaerobiosis was also analyzed. Differences with the pattern obtained from segments incubated anaerobically for 2 h were observed. Previously described rice calcium-dependent protein kinase activities are present in membrane preparations obtained from anaerobically treated roots and coleoptiles. Calcium-dependent phosphorylation of substrates like H1 histone (type III-S) and MARCKS peptide is shown. The rice CDPK activity capable of phosphorylating the MARCKS peptide is two-fold higher in coleoptile membrane preparations than roots.