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

Abstract

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.

Two calcium dependent protein kinases are differently regulated by light and have different activity patterns during seedling growth in Pharbitis nil

“…The obtained results demonstrate that these two CDPKs differ in their temporal and special distribution to accomplish their diverse physiological role. Available data show that most CDPK isoforms are expressed constitutively and neither organ nor tissue specificity were observed (Morello et al 2006). However for some of them a unique, very restricted expression pattern and enzyme activity in different organs or tissues at different stages of growth and development were noted.…”

“…For example, two main enzymes, Suc-phosphate synthase (SPS) and nitrate reductase (NR), are inactivated in the dark by CDPK phosphorylation on specific Ser residues (McMichael et al 1995;Douglas et al 1998). It might coordinate the supply of carbon skeletons and ammonia needed for starch synthesis or amino acids when photosynthesis is off (Cheng et al 2002;Morello et al 2006;Klimecka and Muszyńska 2007).…”

Isolation and analysis of a TIR-specific promoter from poplar

“…It has been demonstrated that enhancer elements are generally present in the introns of the 5′ UTR region, suggesting that the introns located in the 5′ UTR region may play an important role in the regulation of gene expression by regulating mRNA stability, the efficiency of translation, or can directly impact transcriptional regulation (Maas et al, 1991;Gidekel et al, 1996;Bailey-Serres and Gallie, 1998;Plesse et al, 2001;Morello et al, 2002;Fiume et al, 2004;Wang et al, 2004;Gutiér-rez-Alcalá et al, 2005). In addition, plant intron sequences were reported to enhance functional gene expression in transgenic plants, and this intron-mediated enhancement (IME) of gene expression has been reported for several genes, indicating that the first 5′-leader intron is necessary for a high-level gene expression and can autonomously promote transcription (Mascarenhas et al, 1990;Vasil et al, 1990;Maas et al, 1991;Clancy et al, 1994;Rose and Beliakoff, 2000;Kim et al, 2004;Morello et al, 2002Morello et al, , 2006Gutiérrez-Alcalá et al, 2005;Kim et al, 2006;Sivamani and Qu, 2006). Thus, introns may influence and enhance gene expression by multiple mechanisms (Le Hir et al, 2003).…”

Gene expression enhancement mediated by the 5′ UTR intron of the rice rubi3 gene varied remarkably among tissues in transgenic rice plants

“…In several studies, the increase in protein accumulation caused by introns exceeded that in mRNA accumulation, supporting the notion that plant introns also elevate translation (Mascarenhas et al 1990;Bourdon et al 2001;Clancy and Hannah 2002;Rose 2004). Interestingly, IME studies in dicot plants were usually performed in stably transformed plants (Fu et al 1995a, b;Chaubet-Gigot et al 2001;Rose 2002;2004) whereas most IME studies in monocot plants were carried out by transient expression assays in cultured cells (Callis et al 1987;Mascarenhas et al 1990;Rethmeier et al 1997;Bourdon et al 2001;Clancy and Hannah 2002;Morello et al 2006;Sivamani and Qu 2006) with few exceptions (Jeon et al 2000;Dugdale et al 2001;Bourdon et al 2004). Moreover, few studies simultaneously investigated the characteristics of IME in diVerent tissues of plants.…”