Papers by Valérie Pacquit
Plant and Cell Physiology, 1998
Calcium-dependent phosphoeno/pyruvate carboxylase protein kinase was copurified with C 4 phosphoi... more Calcium-dependent phosphoeno/pyruvate carboxylase protein kinase was copurified with C 4 phosphoino/pyruvate carboxylase (C 4 PEPC) from illuminated Sorghum leaves during purification by various procedures. Isolated mesophyll cell protoplasts contained both calcium-dependent and-independent protein kinases. The latter was induced by light and weak bases and was found to be the major protein kinase phosphorylating C 4 PEPC in the mesophyll.
Frontiers in plant science, 2017
Plant non-specific lipid transfer proteins (nsLTPs) belong to a complex multigenic family implica... more Plant non-specific lipid transfer proteins (nsLTPs) belong to a complex multigenic family implicated in diverse physiological processes. However, their function and mode of action remain unclear probably because of functional redundancy. Among the different roles proposed for nsLTPs, it has long been suggested that they could transport cuticular precursor across the cell wall during the formation of the cuticle, which constitutes the first physical barrier for plant interactions with their aerial environment. Here, we took advantage of the Arabidopsis thaliana etiolated hypocotyl model in which AtLTP2 was previously identified as the unique and abundant nsLTP member in the cell wall proteome, to investigate its function. AtLTP2 expression was restricted to epidermal cells of aerial organs, in agreement with the place of cuticle deposition. Furthermore, transient AtLTP2-TagRFP over-expression in Nicotiana benthamiana leaf epidermal cells resulted in its localization to the cell wall,...
European Journal of Biochemistry, 1995
Steady-state kinetic analyses were performed on the non-phosphorylated, in vitro phosphorylated a... more Steady-state kinetic analyses were performed on the non-phosphorylated, in vitro phosphorylated and phosphorylation-site mutant (Ser8→Asp) forms of purified recombinant sorghum C 4 phosphoenolpyruvate (P-pyruvate) carboxylase (EC 4.1.1.3 1) containing an intact N-terminus. Significant differences in certain kinetic parameters were observed between these three enzyme forms when activity was assayed at a suboptimal but near-physiological pH (7.3), but not at optimal pH (8.0). Most notably, at pH 7.3 the apparent K i for the negative allosteric effector l-malate was 0.17 mM, 1.2 mM and 0.45 mM while the apparent K a for the positive allosteric effector glucose 6-phosphate (Glc6P) at 1mM P-pyruvate was 1.3 mM, 0.28 mM and 0.45 mM for the dephosphorylated, phosphorylated and mutant forms of the enzyme, respectively. These and related kinetic analyses at pH 7.3 show that phosphorylation of C 4 P-pyruvate carboxylase near its N-terminus has a relatively minor effect on V and K m (total P-pyruvate) but has a dramatic effect on the extent of activation by Glc6P, type of inhibition by l-malate and, most especially, K a (Glc6P) and K i (l-malate). Thus, regulatory phosphorylation profoundly influences the interactive allosteric properties of this cytosolic C 4-photosynthesis enzyme.
European Journal of Biochemistry, 1992
The light-dependent phosphorylation of the photosynthetic phosphoennlpyruvate carboxylase (PyrPC)... more The light-dependent phosphorylation of the photosynthetic phosphoennlpyruvate carboxylase (PyrPC) was shown to occur in protoplasts from Sorghum mesophyll cells. I t was accompanied by an increase in PyrPC protein-serine-kinase activity and conferred the target-specific functional properties, i. e. an increase in V,,, and apparent Ki for L-malate, as previously found with the whole leaf. The light-dependent regulatory phosphorylation of PyrPC was (a) specifically promoted by the weak bases NH4C1 and methylamine (agents which increase cytosolic pH), but not by KN03, (b) inhibited by the cytosolic protein-synthesis inhibitor, cycloheximide, thus confirming that protein turnover is a component of the signal-transduction cascade, as reported in [4], (c) found to moderately decrease in the presence of EGTA and to be strongly depressed when the Ca2+-selective ionophore A23287 was added to the incubation medium together with EGTA. Addition of Ca2+, but not of Mg2 ', to the Ca2 '-depleted protoplasts partially, but significantly, relieved the inhibition. Calcium deprivation apparently affected the in-situ light-activation of the PyrPC protein kinase. These data indicated that both Ca2+ and an increase in cytosolic pH are required for the induction of PyrPC protein kinase activitylPyrPC phosphorylation in illuminated protoplasts from Sorghum mesophyll cells. In C4 plants, the primary photosynthetic C 0 2 fixation catalysed by phosphoenolpyruvate carboxylase (PyrPC) is a key step through which the control of carbon flow is operated. Regulation of the enzyme, in the cytosol of mesophyll cells, involves both antagonistic effectors i. e. glucose-6P, triose-P (positive), L-malate (negative), [I], and a light-dependent reversible phosphorylation process [2-41. When measured at suboptimal conditions of pH and substrate PyrP (phosphoenolpyruvate) which are supposed to be physiological, the phosphorylated PyrPC displays an increased catalytic activity and Ki for L-malate. This post-translational modulation is thought to represent an additional mechanism allowing the
Photosynthesis Research, 1995
A peptide containing the N-terminal phosphorylation site (Ser-8) of Sorghum C4-phosphoenolpyruvat... more A peptide containing the N-terminal phosphorylation site (Ser-8) of Sorghum C4-phosphoenolpyruvate carboxylase (PEPC) was synthesized, purified and used to raise an antiserum in rabbits. Affinity-purified IgGs prevented PEPC phosphorylation in a reconstituted in vitro assay and reacted with both the phosphorylated and dephosphorylated forms of either native or denatured PEPC in immunoblotting experiments. Saturation of dephospho-PEPC with these specific IgGs resulted in a marked alteration of its functional and regulatory properties that mimicked phosphorylation of Ser-8. A series of recombinant C4 PEPCs mutated in the N-terminal phosphorylation domain and a C3-1ike PEPC isozyme from Sorghum behaved similarly to their C4 counterpart with respect to these phosphorylation-site antibodies.
Australian Journal of Plant Physiology, 1997
A family of synthetic peptides modelled after the highly conserved, N-terminal phosphorylation do... more A family of synthetic peptides modelled after the highly conserved, N-terminal phosphorylation domain of C4 phosphoenolpyruvate carboxylase (PEPC) and a complementary set of recombinant mutant target proteins were exploited to investigate the local structural requirements for phosphorylation of this cytosolic C4 enzyme by its Ca2+-independent protein kinase. The only peptide homolog examined that was significantly phosphorylated by maize (Zea mays L.) leaf PEPC-kinase spanned the P−5 through P+16 region surrounding the target serine residue in maize PEPC (fifth through sixteenth residues on the N- and C-terminal sides, respectively, of the phosphorylatable serine at position ‘P’). However, its apparent Km value was 200-times that of intact C4 PEPC. The results from the related site-directed mutagenesis experiments with the recombinant sorghum (Sorghum vulgare) C4 -enzyme indicated that alteration of several highly conserved residues flanking the target serine with non-conservative A...
Scientific reports, Jan 20, 2016
The rationale of this study is to compare and integrate two heterologous datasets intended to unr... more The rationale of this study is to compare and integrate two heterologous datasets intended to unravel the spatiotemporal specificities of gene expression in a rapidly growing and complex organ. We implemented medium-throughput RNA in situ hybridization (ISH) for 39 genes mainly corresponding to cell wall proteins for which we have particular interest, selected (i) on their sequence identity (24 class III peroxidase multigenic family members and 15 additional genes used as positive controls) and (ii) on their expression levels in a publicly available Arabidopsis thaliana seed tissue-specific transcriptomics study. The specificity of the hybridization signals was carefully studied, and ISH results obtained for the 39 selected genes were systematically compared with tissue-specific transcriptomics for 5 seed developmental stages. Integration of results illustrates the complementarity of both datasets. The tissue-specific transcriptomics provides high-throughput possibilities whereas IS...
Plant Physiology and Biochemistry, 2002
... left double bracket delimiter 19 right double bracket delimiter have reported that the nuclea... more ... left double bracket delimiter 19 right double bracket delimiter have reported that the nuclear factorWIZZ (wound-induced leucine zipper zinc finger) specifically binds to sequences containing two TTGAC core motifs (W box); two identical sequences are present in the lecRK-a1 ...
BMC Plant Biology, 2010
Background: Cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the f... more Background: Cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the final steps in the biosynthesis of monolignols, the monomeric units of the phenolic lignin polymers which confer rigidity, imperviousness and resistance to biodegradation to cell walls. We have previously shown that the Eucalyptus gunnii CCR and CAD2 promoters direct similar expression patterns in vascular tissues suggesting that monolignol production is controlled, at least in part, by the coordinated transcriptional regulation of these two genes. Although consensus motifs for MYB transcription factors occur in most gene promoters of the whole phenylpropanoid pathway, functional evidence for their contribution to promoter activity has only been demonstrated for a few of them. Here, in the ligninspecific branch, we studied the functional role of MYB elements as well as other cis-elements identified in the regulatory regions of EgCAD2 and EgCCR promoters, in the transcriptional activity of these gene promoters.
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Papers by Valérie Pacquit