Journal of Experimental Botany, Vol. 53, No. 373, pp. 1521-1524,
June 2002
© 2002 Oxford University Press
Short Communications |
Cloning and characterization of a phospholipase C from the C4 plant Digitaria sanguinalis
Institut de Biotechnologie des Plantes, UMR 8618, Université Paris XI, Bâtiment 630, 91405 Orsay Cedex, France
Received 5 December 2001; Accepted 11 February 2002
Abstract
As a PLC activity was implicated in the light transduction pathway that controls C4 photosynthesis in Digitaria sanguinalis, a full length PLC cDNA (DsPLC2) was cloned. The proteins encoded by the two possible open reading frames were produced in Escherichia coli; they both harbour a PLC activity but with different response to Ca2+ concentration, and with different sensitivity to the PLC inhibitor U-73122.
Key words: Monocot plant, phosphoinositide-specific phospholipase C, recombinant protein, translation initiation.
Introduction
In C4 plants such as maize and sorghum, the primary step in photosynthesis involves the CO2 fixation by a specific isoform of phosphoenolpyruvate carboxylase (C4 PEPC). This enzyme is regulated by a reversible, light-dependent phosphorylation process (Vidal and Chollet, 1997
). A PLC was suggested to be involved in the transduction cascade leading to C4 PEPC phosphorylation in mesophyll cells from the C4 monocot plant, D. sanguinalis (Coursol et al., 2000).
In various signal transduction pathways, PLC (EC 3.1.4.11) catalyses the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to yield inositol 1,4,5-trisphosphate and 1,2-diacylglycerol, both of which act as second-messengers in cell signalling (Berridge, 1993). In animals, four different classes of PLC termed ß, 
, 
, and 
, have been described (Lopez et al., 2001); their regulation involves GTP-binding proteins or tyrosine kinases. All the PLC isoforms contain two regions of high sequence homology, X and Y, which constitute the enzyme's catalytic domain, and a C2-like domain. In addition, other accessory modules, a pleckstrin homology (PH) domain and an EF-hand domain are also present in some PLC isoforms (Katan, 1998). In plants, PLCs identified so far, are strictly dependent on Ca2+ for activity like the other eukaryotic PLCs; they are more closely related to the animal isotype (Munnik et al., 1998), however, they do not possess the PH domain. To date, little is known about the regulatory mechanism acting on the plant enzymes and their implication in signal transduction pathways.
In order to study in more detail the role of PLC in the control of C4 PEPC phosphorylation in C4 plants, a full-length PLC cDNA was isolated from D. sanguinalis. The deduced protein sequence contains the conserved catalytic domains of PLCs. In this paper, in vitro activity tests on recombinant proteins synthesized from the two potential initiation codons identified in the cDNA, were reported.
Materials and methods
Plant material
The plant material used throughout the present study was leaves harvested from 6-week-old plants of Digitaria sanguinalis (L.) and mesophyll cell protoplasts prepared from these leaves (Giglioli-Guivarch et al., 1996).
Cloning of DsPLC2 cDNA
Full length PLC cDNA from D. sanguinalis was obtained using 5'- and 3'-rapid amplification of cDNA ends (RACE). A 600 bp D. sanguinalis DNA fragment located in the X domain of a PLC gene, was first cloned using degenerate primers (5'-TCTCGAGACIGGICAYAAYTCHTA-3' and 5'-CGATATCYTTWGGIGGYTTDGTTG-3' corresponding to TGHNSY and STKPPE peptides, respectively) deduced from regions with high sequence homology in different plant PLCs. First strand cDNA synthesis was carried out using 2 µg of poly(A+) mRNA, the modified lock-docking oligo(dT) primer provided with the Marathon cDNA amplification kit (Clontech) and the Superscript II reverse transcriptase (Life Technologies). Second strand synthesis was achieved using the Marathon cDNA amplification kit and following the manufacturer's instructions. For RACE reactions, ELONGASE Enzyme Mix (Life Technologies), Marathon cDNA adaptor primer and a gene-specific primer were used. The gene-specific primers for RACE amplification were as follows: 5'-CTGACCGGTAATCAACTCAGCAG-3' for 3'-RACE, and 5'-GGAAATTCTTGAAGATGTTTTGATTCAG-3' for 5'-RACE. The PCR products of the 5'- and 3'-RACE, respectively, were cloned into pBluescript II SK (+) and sequenced using the ABI automated DNA sequencer (Applied Biosystem).
RT-PCR
Total RNA (5 µg) was reverse-transcribed into first strand cDNA at 42 °C for 1 h, with the PLC-specific reverse primer, 5'-CGATCTCAGGGACAGTCAG-3' hybridizing 170 bp upstream of the stop codon, and 200 units of reverse transcriptase (Supercript II, Life Technologies) in a 20 µl reaction. PCR amplification was performed on 2 µl of a one-tenth dilution of the first strand cDNA reaction using the two following primers: 5'-GTTCTCCATGGAAGGACAC-3' and 5'-GGTACCACCCTTTGCTTC-3'.
Expression, purification, and characterization of His-DsPLC2 fusion proteins in E. coli
DsPLC2 cDNA was cloned into the expression vector pQE-30 (Qiagen) by ligation of 5'- and 3'-moieties of the cDNA obtained after PCR amplification by the proof reading Pfu DNA polymerase (Stratagene) using the 5'- and 3'-RACE clones as templates. Two amplifications of the 5'-part of the cDNA were performed in order to clone the coding sequence from the first and the second ATG of the cDNA open reading frame. The cloning strategy preserved the native stop codon of DsPLC2. The resulting plasmids denominated His-PLC2 and His-PLC2
allow the synthesis of a 630 and 690 amino acids sequence of DsPLC2, respectively, fused to a sequence of 12 amino acids containing a 6x His tag. These plasmids were used to transform E. coli BL21 containing a pREP4 KanR plasmid which carries the lacI gene encoding the lac repressor. The 6x His recombinant proteins were produced in E. coli and purified using the TALONTM IMAC Resin (Clontech) following the instructions of the manufacturer.
Proteins were separated electrophoretically on SDS-PAGE and transferred to nitrocellulose membranes. Recombinant PLC proteins were probed either with INDIATM HisProbe-HRP (Pierce) or a polyclonal antibody raised against potato PLC (kindly provided by Dr C Pical, Lund University, Lund, Sweden).
PLC activity was assayed according to Coursol et al. (Coursol et al., 2000), except that the non-radioactive substrate was a synthetic PIP2 (Echelon). No PLC activity was detected in protein extracts from E. coli without the recombinant plasmids.
Results and discussion
Cloning and sequence analysis of DsPLC2 cDNA
Degenerate oligonucleotide primers designed from two conserved regions located in the X domain of plant PLC amino acid sequences were used for PCR amplification with D. sanguinalis DNA. The PCR-amplified fragment (600 bp) encodes an amino acid sequence homologous to the X domain of PLCs and contains two introns at the same positions as those identified in the Arabidopsis ATHAPLC1G gene (accession No. U76 423; Hartweck et al., 1997).
Based on the sequence of the D. sanguinalis PLC-like DNA fragment, primers were designed to perform a rapid amplification of cDNA ends. Three cDNA encoding putative PLCs were identified, DsPLC1 for which only the 5'-end has been isolated, a DsPLC1 variant for which an intron located in the X domain sequence was not spliced due to a mutation in the 5'-splicing site (leading to a predicted truncated polypeptide), and DsPLC2 for which both ends have been cloned.
The full length DsPLC2 cDNA (EMBL accession No. AJ291467) consists of 2013 bp upstream of the poly(A+) tail, which includes a 1890 bp open reading frame. An in-frame stop codon was present at nucleotides -19 to -21 upstream of an ATG codon, suggesting that this ATG is the start codon (Fig. 1). The corresponding predicted amino acid sequence is 630 amino acids long with a calculated molecular mass of 70.8 kDa. DsPLC2 was identified as a putative PLC since it contains the catalytic domain composed of the X (including two conserved His residues) and Y regions, and the C2-like domain found in other PLCs. The N-terminal region of DsPLC2 does not contain obvious subcellular sorting signals or transmembrane-spanning domains and therefore this enzyme is most likely located in the cytosol. Two helix-loop-helix motifs located upstream of the X domain (the corresponding four 
-helices are indicated in Fig. 1) were predicted using the SWISS-MODEL Protein Modelling Server (Guex et al., 1999); they would correspond to the second lobe of the EF-hand domain of PLC-1 (Essen et al., 1996).
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The primary structure of DsPLC2 is similar to that of currently known plant PLC homologues. The DsPLC2 deduced amino acid sequence exhibits 66% overall sequence identity to Oryza sativa predicted protein (the only monocot PLC sequence available to date besides DsPLC2, AF332874); this identity is 83% within the X domain, 84% within the Y domain, and 79% within the C2-like domain.
Alignment of known plant PLC sequences (originating from C3 dicot plants with the exception of PLC from O. sativa, a C3 monocot) with DsPLC2 (C4 monocot) shows that the initiating methionine in these PLCs maps at or near the second methionine found in DsPLC2. Thus, DsPLC2 could have a unique structure with an additional 40 amino acid stretch at the enzyme's N-terminus. The presence of two in-frame initiation codons in DsPLC2 cDNA raises the question of which one is actually used as a translation start site, especially as the flanking nucleotide sequence around these two putative translational initiation sites fulfils the Kozak criteria for initiation (Kozak, 1987).
RT-PCR for mRNA expression studies
Since PLC activity has been implicated as an early event in the light induction process of C4 photosynthesis in D. sanguinalis, the occurrence of DsPLC2 mRNA was analysed by RT-PCR in extracts from illuminated leaves and induced mesophyll protoplasts. Two gene-specific primers annealing sequences in exons flanking the second intron identified in the D. sanguinalis PLC DNA fragment were used for amplification, in order to differentiate amplification from cDNA and from contaminating genomic DNA. Furthermore, the forward primer was chosen to overlap the first intron identified in this same DNA fragment to avoid amplification from the unspliced cDNA.
As shown in Fig. 2, DNA bands corresponding to the expected sizes (296 bp for amplification from cDNA and 369 bp for amplification from DNA) and hybridizing to DsPLC2 cDNA probe, were obtained in the samples examined. This demonstrates that DsPLC2 gene is expressed in leaves and mesophyll protoplasts.
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Recombinant DsPLC2 fusion proteins possess PLC activity
To ascertain that the cloned DsPLC2 cDNA encodes a functional enzyme, His-fusion proteins for the two possible reading frames (His-PLC2 and His-PLC2 for His-tag fusion to the first and the second ATG, respectively) were produced in E. coli. Soluble recombinant proteins (most of the fusion proteins were found in the insoluble fraction) were purified on TALON metal affinity resin and elution fractions were analysed by blotting and activity.
The two His-tagged recombinant proteins, His-PLC2 and His-PLC2 (72 and 68 kDa expected molecular masses, respectively) were both detected by the His-probe (Fig. 3A) as polypeptides of about 75 kDa apparent molecular mass (in our experimental conditions, the proteins differing by only 4 kDa were not resolved by SDS-PAGE). A polypeptide with this same electrophoretic mobility was also revealed in both elution fractions by a potato PLC-antibody (Fig. 3A).
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His-PLC2 and His-PLC2
displayed PLC activity. The results (Fig. 3B
) show that whereas the maximum activity of PIP2 hydrolysis (similar for the two proteins) was observed at 1 µM Ca2+ for His-PLC2, it was reached at 10 µM Ca2+ for His-PLC2. The aminosteroid inhibitor U-73122 has been used widely to study PLC-dependent processes. When added (at a 30 or 50 µM concentration) to the assay, U-73122 reduced PLC activity of the recombinant proteins (40% and 20% inhibition for His-PLC2 and His-PLC2, respectively; Fig. 3C) whereas the inactive analogue U-73122, had no effect (Fig. 3C). These results show that whatever the ATG start codon used, DsPLC2 cDNA encodes a polypeptide with PLC activity. However, the two DsPLC2 recombinant proteins displayed significantly different Ca2+ requirements and U-73122 sensitivity. The functional identity of DsPLC2 cDNA was thus established, however, whether the two translation start codons are used in mesophyll cells, remain to be clarified.
Acknowledgments
We thank Dr A Tremolière's group for stimulating discussions and Dr C Pical (DPB, Lund University, Lund, Sweden) for generous gift of the potato PLC-antibody and sharing unpublished results.
Notes
1 Present address: Biology Department, Pennsylvania State University, 208 Mueller Laboratory, University Park, PA 16802-5301, USA. 
2 To whom correspondence should be addressed. Fax: +33 1 69 15 34 23. E-mail: Jean.Vidal{at}ibp.u-psud.fr
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