Plants possess a large set of transcription factors both involved in the control of plant development or in plant stress responses coordination. We previously identified PRR2, a Pseudo-Response Regulator, as a plant-specific CML-interacting partner. We reported that PRR2 acts as a positive actor of plant defense by regulating the production of antimicrobial compounds. Here, we report new data on the interaction between PRR2 and transcription factors belonging to the Teosinte branched Cycloidea and PCF (TCP) family. TCPs have been described to be involved in plant development and immunity. We evaluated the ability of PRR2 to interact with seven TCPs representative of the different subclades of the family. PRR2 is able to interact with TCP13, TCP15, TCP19 and TCP20 in yeast two-hybrid system and in planta interactions were validated for TCP19 and TCP20. Transient expression in tobacco highlighted that PRR2 protein is more easily detected when co-expressed with TCP19 or TC20. This stabilization is associated with a specific sub-nuclear localization of the complex in Cajal bodies or in nuclear speckles according to the interaction of PRR2 with TCP19 or TCP20 respectively. The interaction between PRR2 and TCP19 or TCP20 would contribute to the biological function in specific nuclear compartments.

Calcium signalling mediated by Calmodulin (CaM) and calmodulin-like (CML) proteins is critical to plant immunity. CaM and CML regulate a wide range of target proteins and cellular responses. While many CaM-binding proteins have been identified, few have been characterized for their specific role in plant immunity. Here, we report new data on the biological function of a CML-interacting partner, PRR2 (PSEUDO-RESPONSE REGULATOR 2), a plant specific transcription factor. Until now, the physiological relevance of PRR2 remained largely unknown. Using a reverse genetic strategy in A. thaliana, we identified PRR2 as a positive regulator of plant immunity. We propose that PRR2 contributes to salicylic acid (SA)-dependent responses when challenged with the phytopathogenic bacterium Pseudomonas syringae. PRR2 is transcriptionally upregulated by SA and P. syringae, enhances SA biosynthesis and SA signalling responses; e.g. in response to P. syringae, PRR2 induces the production of SA and the accumulation of the defence-related protein PR1. Moreover, PRR2 overexpressing lines exhibit an enhanced production of camalexin, a phytoalexin that confers enhanced resistance against pathogens. Together, these data reveal the importance of PRR2 in plant immune responses against P. syringae and suggest a novel function for this particular plant specific transcription factor in plant physiology.

A clone for a novel Arabidopsisthaliana calmodulin (CaM)-binding protein of 25 kDa (AtCaMBP25) has been isolated by using a radiolabelled CaM probe to screen a cDNA expression library derived from A. thaliana cell suspension cultures challenged with osmotic stress. The deduced amino acid sequence of AtCaMBP25 contains putative nuclear localization sequences and shares significant degree of similarity with hypothetical plant proteins only. Fusion of the AtCaMBP25 coding sequence to reporter genes targets the hybrid protein to the nucleus. Bacterially expressed AtCaMBP25 binds, in a calcium-dependent manner, to a canonical CaM but not to a less conserved isoform of the calcium sensor. AtCaMBP25 is encoded by a single-copy gene, whose expression is induced in Arabidopsis seedlings exposed to dehydration, low temperature or high salinity. Transgenic plants overexpressing AtCaMBP25 exhibits an increased sensitivity to both ionic (NaCl) and non-ionic (mannitol) osmotic stress during seed germination and seedling growth. By contrast, transgenic lines expressing antisense AtCaMBP25 are significantly more tolerant to mannitol and NaCl stresses than the wild type. Thus, the AtCaMBP25 gene functions as a negative effector of osmotic stress tolerance and likely participates in stress signal transduction pathways.

A cDNA clone (LeCRK1), encoding a novel isoform of calcium-dependent protein kinase (CDPK), was isolated by screening a tomato (Lycopersicon esculentum) cDNA library. The protein derived from the full-length sequence indicated that it belongs to the family of CDPK-related kinases (CRKs) and the predicted amino acid sequence shows a modular organization of the protein consisting of different characteristic domains. The kinase domain of LeCRK1 shares a high degree of similarity with the catalytic domain of CDPKs. In contrast to canonical members of the family, LeCRK1 has a degenerate sequence in the C-terminal calmodulin-like domain. LeCRK1 protein was shown to be a functional kinase, but, consistent with the lack of calcium-binding activity, its autophosphorylation activity did not require calcium. LeCRK1 harbours an amphiphilic amino acid region revealed to be a functional calmodulin-binding site by in vitro assay. A putative myristoylation/palmitoylation sequence has been identified at the N-terminus. Expressing an LeCRK1::GFP fusion protein in the protoplast resulted in its targeting to the plasma membrane. Site-directed mutagenesis of critical amino acids of the myristoylation/palmitoylation consensus sites led to the accumulation of the mutated protein in the cytoplasm, suggesting that the native protein is anchored to the plasma membrane by acylated residues. Expression studies revealed significant accumulation of LeCRK1 transcripts during fruit ripening, although transcripts were also detected in stem, leaf, and flower. LeCRK1 mRNA level in leaves was slightly induced by ethylene and salicylic acid, and upon mechanical wounding and cold treatment. It is noteworthy that LeCRK1 mRNAs were undetectable in different tomato-ripening natural mutants such as NR, Rin, and Nor, suggesting a role in the ripening process.

Screening a cDNA expression library with a radiolabelled calmodulin (CaM) probe led to the isolation of AtCaMRLK, a receptor-like kinase (RLK) of Arabidopsis thaliana. AtCaMRLK polypeptide sequence shows a modular organization consisting of the four distinctive domains characteristic of receptor kinases: an amino terminal signal sequence, a domain containing seven leucine-rich repeats, a single putative membrane-spanning segment and a protein kinase domain. Using truncated versions of the protein and a synthetic peptide, we demonstrated that a region of 23 amino acids, located near the kinase domain of AtCaMRLK, binds CaM in a calcium-dependent manner. Real-time binding experiments showed that AtCaMRLK interacted in vitro with AtCaM1, a canonical CaM, but not with AtCaM8, a divergent isoform of the Ca2+ sensor. The bacterially expressed kinase domain of the protein was able to autophosphorylate and to phosphorylate the myelin basic protein, using Mn2+ preferentially to Mg2+ as an ion activator. Site-directed mutagenesis of the conserved lysine residue (Lys423) to alanine, in the kinase subdomain II, resulted in a complete loss of kinase activity. CaM had no influence on the autophosphorylation activity of AtCaMRLK. AtCaMRLK was expressed in reproductive and vegetative tissues of A. thaliana, except in leaves. Disruption in the AtCaMRLK coding sequence by insertion of a DsG transposable element in an Arabidopsis mutant did not generate a discernible phenotype. The CaM-binding motif of AtCaMRLK was found to be conserved in several other members of the plant RLK family, suggesting a role for Ca2+/CaM in the regulation of RLK-mediated pathways.

Phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinases in animals, elicits the transient activation of a 45-kDa protein kinase in tobacco cell-suspension cultures. The 45-kDa protein kinase preferentially phosphorylates myelin basic protein (MBP), a general substrate for MAPK. Studies using cycloheximide indicated that protein synthesis is not required for the activation of the kinase. Treatment of tobacco cell extracts containing the activated kinase with either serine/threonine-specific or tyrosine-specific protein phosphatase abolished the kinase activity, which consequently appears to be regulated by phosphorylation. By using an immune complex kinase assay with antibodies specific for stress-responsive MAPKs, we show that the PMA-activated kinase is immunologically related to the wound-induced protein kinase (WIPK), and not to the salicylic acid-induced protein kinase (SIPK), two representative members of the tobacco MAPK family, known to be activated by extracellular stimuli. Furthermore, the activated kinase was recognized by phospho-specific MAPK antibodies. Collectively, these results indicate that phorbol ester promotes the activation of a 45-kDa protein kinase related to WIPK in tobacco cells. Activation of WIPK in response to PMA is associated with protein phosphorylation but not with an increase in protein level.

Water‐soaking, a physiological disorder characterised by a glassy texture of the flesh, depreciates greatly the commercial quality of early‐season Charentais cantaloupe melons (Cucumis melo L. cv. Talma). Although it is accepted that the genotype and a number of physiological and environmental factors play a role in the development of the syndrome, the intimate mechanisms responsible for water‐soaking remain unknown. We report here on an integrated study of the development of water‐soaking in fruit. Using nuclear magnetic resonance (NMR) imaging, we have shown that water mobility increased in the diseased tissues. Alteration of the cell wall and the presence of large intercellular spaces were correlated with a severe depletion of cell wall calcium. Water‐soaking developed during the late stages of fruit ripening, but no correlation was found with ethylene biosynthesis. Thus, fruits in which ethylene action was blocked by 1‐methylcyclopropene remained sensitive to water‐soaking. Moreover, the expression of two genes encoding key enzymes in ethylene biosynthesis remained unchanged in response to water‐soaking. The major changes observed concerned a protein implicated in calcium signalling processes. While the amount of total calmodulin, the ubiquitous calcium binding protein, was not modified, a particular calmodulin‐binding protein (CaM‐BP) was absent in water‐soaked but not in sound mature tissues. This CaM‐BP may be a marker or a determinant of this physiological disorder.

Tobacco cell suspension cultures respond to treatment with phorbol 12-myristate 13-acetate (PMA), by the accumulation of transcripts for hsr203J, a plant defence gene. We provide evidence that the effect of PMA on hsr203J gene expression depends upon a transient production of active oxygen species (AOS), an early response of the cell suspensions to PMA. Pre-treatment with diethyldithiocarbamate (DDC), an inhibitor of superoxide dismutase (SOD), abolishes the PMA-induced generation of AOS and the subsequent expression of the hsr203J gene. Conversely, a direct application of hydrogen peroxide to the cell suspensions causes an increase in hsr203J transcripts level, indicating that AOS such as hydrogen peroxide act as signalling molecules to induce the expression of the hsr203J gene. Collectively, these data establish that AOS are critical intermediates mediating PMA action on hsr203J gene expression.

Hsr203J, a tobacco gene associated with the hypersensitive response to Ralstonia solanacearum, presents an novel pattern of activation: high, rapid, local and specific activation in response to HR-inducing bacterial isolates. The transcriptional regulation of this gene has been extensively studied: a 28 bp regulatory element, necessary and sufficient for specific activation has been identified, which is involved in the formation of an HR-inducible DNA/protein complex. In addition, the expression product of hsr203J has esterase activity. Finally, the biological role of hsr203J has been estimated using sense and antisense strategies, and the results suggest that the gene product plays at role as a negative regulator of the HR.

We have recently reported the isolation of a tobacco gene, hsr 203J, whose transcripts accumulate during the hypersensitive reaction, a plant response associated with resistance to pathogens. We present and discuss here some structural and biochemical properties of the gene product. Nucleotide sequence analysis has shown that the hsr 203J gene contains an open reading frame coding for a polypeptide of 335 amino acids. The predicted amino acid sequence contains the GXSXG motif characteristic of serine hydrolases, and displays limited but significant similarity to lipases and esterases of prokaryotic origin. The hsr 203J gene was expressed in Escherichia coli, and the recombinant protein, purified to near homogeneity, was able to degrade p-nitrophenylbutyrate, a general substrate for carboxylesterases. The enzyme was unable to hydrolyze lipids, and was active on short-chain acyl esters only. The hydrolytic activity was abolished by diisopropyl fluorophosphate and a derivative of isocoumarin, as expected for a member of the serine hydrolase family. Sequence similarities between the tobacco esterase and expressed sequence tags in databases suggest the existence of members of this enzyme family in various plant species.

A bean cDNA clone that specifies a 70 kDa heat-shock protein (hsp70) has been isolated and sequenced. The nucleotide sequence analysis shows that the cDNA could encode a 72 kDa protein that is highly related to prokaryotic and mitochondrial members of the hsp70 family. The predicted protein was found to contain an amino-terminal extension typical of transit sequences. The in vitro transcription/translation product of the cDNA behaved as a 72 kDa polypeptide as predicted from the longest open reading frame. This polypeptide could be imported into isolated mitochondria and recovered as a 68 kDa product. The imported protein is identical in size to a mitochondrial protein that cross-reacts with hsp70-specific antibodies. The import data and Western blot analysis suggest that the cDNA clone encodes a mitochondrial member of the hsp70 family. Electrophoretic and immunoblot analysis reveal that the protein is loosely associated to the mitochondrial envelope and also exists as discrete soluble protein aggregates of about 270 and 420 kDa. Hsp70 of bean mitochondria can be in vitro phosphorylated on threonine residues in a calcium-dependent manner, and the modified protein was detected as an oligomer of about 160 kDa only. The data are discussed with respect to the chaperone function of hsp70 in mitochondria.

Crystals of the hexadecameric form of ribulose-bisphosphate carboxylase used to solve the structure of the enzyme are composed of protein substantially crosslinked by a disulfide bond between pairs of large subunits. Conditions leading to the selective formation of dimers of the large subunits are described. The stability and specificity of the intra-dimeric crosslink was used to confirm that only one cysteine residue, Cys247 of neighboring large subunits, is involved in the bridge. The ability to generate this disulfide selectively, or alternatively replace the cysteine by site-directed mutagenesis, has led us to conclude that there is no effect of these changes on any of the critical kinetic parameters of the enzyme. The benign effect of the oxidation indicates that the crystal structures of the ribulose-bisphosphate carboxylase, particularly of the active site, are a true representation of the native enzyme.

Truncations of the subunit of ribulose bisphosphate carboxylase/oxygenase (Rubisco) from Rhodospirillum rubrum were generated by site‐directed mutagenesis to examine the role of the C‐terminal tail section. Removal of the last and the penultimate alpha‐helices in the tail section changes the quaternary structure of the protein. Electrophoretic and electron microscope analysis revealed that the truncated subunits assemble into an octamer, whereas the wild‐type enzyme has a dimeric structure. The octomerization of the mutant protein is due to a hydrophobic patch exposed to the solvent by truncation of the subunit. The mutant protein thus consists of four dimers, bound end‐to‐end by hydrophobic interactions. Insertion of a polar amino acid in the hydrophobic patch by a L424 to N424 substitution restores the familiar dimeric structure. Truncation of the subunit is associated with a considerable decrease in catalytic activity. The mutants undergo carbamylation but bind the reaction intermediate analog, 2‐carboxy arabinitol‐1,5‐bisphosphate, poorly. This indicates that loss of activity in the mutant is due to weakened substrate binding. These findings suggest that the mutations in the tail section of the subunit are transmitted to the active site, although the C‐terminal region is far from the active site. On the basis of the crystal structure of Rubisco, we propose a model for how the truncations of the enzyme subunit induce conformational changes in one of the two phosphate binding sites.

Recent progress in in vitro genetic manipulations (1), and in the structural analysis of Rubisco (2,3) have provided the basis for a rationale mutagenesis of this key enzyme in the photosynthetic carbon metabolism, in attempts to define structure/function relationships. In combination with a better knowledge in the chemistry of the enzymatic reactions, studies of specific changes of highly conserved residues within the active site have been developed. So far, the role of at least two residues essential for activation (Lys 191 in Rhodospirillum rubrum Rubisco) or catalysis (Lys 166) have been defined (4,5). Another strategy consists to examine the functional importance of peptide regions of low homology. Construction of chimaeric genes by sequence replacement have indicated the critical requirement of some regions of the large subunit (i.e. N-terminus, bridge region between N- and C-terminal domains) for the assembly and/or function of the protein (6,7). A similar approach was used here to investigate the role of the C-terminus of the large subunit. Sequence deletion in the tail domain was performed on the gene coding for R.rubrum Rubisco. Mutation was designed to remove the last and pen-ultimate α-helices from the C-terminal extension (Fig.l).

Mesophyll cells were isolated from sunflower leaves by an enzymic procedure. The cell suspensions possessed high photosynthesis rates. The products of cell photosynthesis were similar to the products of leaf disc photosynthesis. The relatively high radioactivity incorporated into malate after (14)CO2 feeding suggests that PEP carboxylase might participate in CO2 fixation. Sunflower leaf extracts possessed a PEP carboxylase activity slightly higher than that of other C3 species. Inhibition of PEP carboxylase by maleate decreased cell photosynthesis by only 15% and the first products of cell photosynthesis were phosphorylated compounds. It is concluded that the high photosynthesis rates displayed by sunflower are not due to a parallel C4 pathway of photosynthesis but are rather dependent, at least in part, on the activity, or the amount, of RuBP carboxylase.

Extracts from sunflower leaves possess a high ribulose-1,5-bisphosphate (RuBP) carboxylase capacity but this enzyme activity is not stable. A purification procedure, developed with preservation of carboxylase activity by MgSO4, yielded purified RuBP carboxylase with high specific activity (40 nkat mg(-1) protein). Measurement of kinetic parameters showed high Km values (RuBP, HCO 3 (-) ) and high Vmax of the reaction catalyzed by this sunflower enzyme; the results are compared with those obtained for soybean carboxylase. Enzyme characteristics are discussed in relation to stabilization and activation procedures and to the high photosynthesis rates of this C3 species.