JXB Advance Access originally published online on April 8, 2004
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Journal of Experimental Botany, Vol. 55, No. 401, pp. 1437-1439, June 1, 2004
© 2004 Oxford University Press
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Isolation of cDNAs encoding typical and novel types of phosphoinositide-specific phospholipase C from the moss Physcomitrella patens
Received 3 December 2003; Accepted 26 February 2004
1 National Institute for Basic Biology, 38 Nishigonaka, Myodaiji, Okazaki 444-8585, Japan
2 Institut für Biologie–Pflanzenphysiologie, Freie Universität Berlin, Königin-Luise-Str. 12-16, D-14195 Berlin, Germany
* To whom correspondence should be addressed. Fax: +81 564 54 4866. E-mail: mikami{at}nibb.ac.jp
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Abstract |
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Two cDNAs encoding proteins, PpPLC1 and PpPLC2, with catalytic and C2 domains conserved in plant phosphoinositide-specific phospholipase C (PI-PLC) were isolated from Physcomitrella patens. The N domain, which has been identified in Arabidopsis PI-PLCs as an EF hand-like domain, was found in both isoforms, although that in PpPLC2 was a split type. At micromolar Ca2+ concentrations, PpPLC1 preferentially hydrolysed phosphatidylinositol-4,5-bisphosphate, while PpPLC2 showed no specificity. Furthermore, at millimolar Ca2+, phosphatidylinositol was hydrolysed by PpPLC2, but not by PpPLC1. Thus, PpPLC1 and PpPLC2 are typical and novel types of plant PI-PLC, respectively.
Key words: s: Phosphoinositide-specific phospholipase C, Physcomitrella patens, substrate preference.
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Phosphoinositide-specific phospholipase C (PI-PLC) hydrolyses phosphatidylinositol-4,5-bisphosphate (PIP2), which results in the generation of inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DG), and both act as second messengers in mammals (Rhee, 2001). Genes encoding plant PI-PLCs, which were proposed to be involved in the responses to light, osmotic stress, gravity, pathogen attack, abscisic acid (ABA), and auxin (Meijer and Munnik, 2003), have already been isolated from various species to date (Hirayama et al., 1995; Shi et al., 1995; Kopka et al., 1998; Otterhag et al., 2001; Coursol et al., 2002; Hunt et al., 2003). PI-PLC isoforms in mammals have been divided into four classes, ß-,
-, 
-, and 
-types, all of which contain the catalytic domain that consists of X and Y regions and regulatory domains such as pleckstrin homology (PH), EF-hand and C2 domains (Rhee, 2001). By contrast, plant PI-PLCs analysed so far all show -type organization but lacking the PH and typical EF-hand domains (Meijer and Munnik, 2003). Since the PH and EF-hand domains are responsible for membrane-localization and Ca2+-binding in mammals (Rhee, 2001), the mode of activation of plant PI-PLCs is probably different from those in mammals. However, little is known about the molecular mechanisms of the activation of plant PI-PLCs.
The moss Physcomitrella patens is now recognized as a model system for plants with the easy application of molecular genetic approaches such as gene-targeted mutagenesis via the homologous recombination (Schaefer, 2002). Searching of the plant EST databases with BLAST algorithm against AtPLC1S from Arabidiopsis thaliana revealed four Physcomitrella cDNA clones, PPU141114, PPU140521, PPU070504, and PPU161218 (GenBank accession numbers are AW561394
[GenBank]
, AW561280
[GenBank]
, AW496918
[GenBank]
, and AW599541
[GenBank]
, respectively), all of which were obtained from ‘The Physcomitrella EST Programme’ (University of Leeds, UK, and Washington University, St Louis, USA). Sequencing analysis indicated that PPU141114 contained the long insert of 1384 bp that comprised two partial cDNAs for PI-PLC (908 bp) and 
-tubulin (468 bp) with poly(A) tails, both of which were connected by the SacI-linker of 8 bp in head-to-head orientation, while PPU070504 contained a short cDNA fragment of 653 bp with a poly(A) tail that was overlapped to PPU141114 without any base changes. The 5'-RACE and the subsequent 3'-RACE reactions resulted in the isolation of a corresponding full-length cDNA of 2424 bp containing an ORF for a PI-PLC homologue of 633 amino acids and calculated molecular mass of 70.8 kDa, which was designated PpPLC1 (accession number AB114834
[GenBank]
). The full-length cDNA did not contain any part of the ORF for -tubulin, thus PPU141114 arrows as an artefact during preparation of the EST library. Although PPU140521 was not analysed because of the severe contamination with other ESTs in the clone obtained, the nucleotide sequence of the EST deposited in the databases was identical to that of a corresponding region of the PpPLC1 cDNA. In addition, using the sequence of PPU161218, a corresponding 2423 bp full-length cDNA, which contains an ORF for a PI-PLC homologue of 639 amino acid and has a calculated molecular mass of 71.8 kDa and is designated PpPLC2 (accession number AB117760
[GenBank]
) was isolated by the 5'-RACE reaction.
As shown in Fig. 1, the overall structure of PpPLC1 and PpPLC2 was similar to those of known plant PI-PLCs comprising the catalytic domain and the C2 domain (Meijer and Munnik, 2003). In addition, the N domain (Otterhag et al., 2001), a regulatory domain with structural similarity to the second loop of the EF-hand domain of rat PLC1, was found in the N-terminal extensions in PpPLC1 and PpPLC2, although there was an insertion in the N domain of PpPLC2 (Fig. 1). Since the split N domain is encoded by a single exon in the PpPLC2 gene (data not shown), the alternative splicing is not responsible for making such an insertion.
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In the X and Y regions, 11 amino acids have been identified as essential residues for PI-PLC activity via binding to Ca2+ and substrate (Essen et al., 1996). It was found that all of them are well conserved in PpPLC1, although a conserved serine residue was replaced by an asparagine residue at amino acid position 409 in PpPLC2 (data not shown). In fact, PLC-like proteins with amino acid substitutions of residues important for PLC activity have already been isolated from rat and human, which have no PIP2-dependent PLC activity (Kanematsu et al., 1996; Otsuki et al., 1999). These findings suggest that PpPLC2 has no PIP2-hydrolysing activity.
To address this possibility, the in vitro activity of recombinant His-tagged PpPLC1 and His-tagged PpPLC2, whose purity had been visually confirmed by SDS-PAGE (data not shown), was examined. As shown in Fig. 2A, His-tagged PpPLC1 hydrolysed PIP2 with a maximum activity of 38 nmol min–1 mg–1 of protein at a physiological concentration of Ca2+ around 10 µM and lower activity at higher Ca2+ concentrations, consistent with other recombinant plant PLCs (Hirayama et al., 1995; Shi et al., 1995; Kopka et al., 1998; Otterhag et al., 2001; Coursol et al., 2002; Hunt et al., 2003). By contrast, His-tagged PpPLC2 hydrolysed PIP2 with very low activity under the same conditions (Fig. 2B). When phosphatidylinositol (PI) was used as a substrate in the reactions with various concentrations of Ca2+, both His-tagged PpPLC1 and His-tagged PpPLC2 showed a very low hydrolysing activity of 1.3 and 9.4 nmol min–1 mg–1 of protein, respectively, at 10 µM Ca2+ (Fig. 2A, B). However, at 1 mM Ca2+, His-tagged PpPLC2 hydrolysed PI very efficiently (22 nmol min–1 mg–1 of protein) than His-tagged PpPLC1 (3.5 nmol min–1 mg–1 of protein) (Fig. 2A, B). Together with their structural characteristics, it is concluded that PpPLC1 and PpPLC2 are typical and novel types of plant PLC, respectively.
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During the review process of this manuscript, a cDNA encoding PI-PLC without PIP2-hydrolysing activity was isolated from pea (Venkataraman et al., 2003). Since the pea PI-PLC contains the typical N-domain and no substitution of the conserved amino acid residues in the catalytic domain, the mechanisms of discrimination against PIP2 are probably different between PpPLC2 and pea PI-PLC.
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Acknowledgements |
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We wish to thank ‘The Physcomitrella EST Programme’ at the University of Leeds (UK) and Washington University, St Louis (USA) for kindly supplying Physcomitrella EST clones. We also thank Professor Jon Hughes (Institut für Pflanzenphysiologie, Justus-Liebig-Universität Giessen, Germany) for critical reading of the manuscript and Sabine Buchert and Cornelia Görick for skilful technical assistance. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB429).
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