SummaryMany stimuli, such as hormones and abiotic stress factors, elicit changes in intracellular calcium levels that serve to convey information and activate appropriate responses. The Ca 2+ signals are perceived by different Ca 2+ receptors, and calmodulin (CaM) is one of the best-characterized Ca 2+ sensors in eukaryotes. Calmodulinlike (CML) proteins also exist in plants; they share sequence similarity with the ubiquitous and highly conserved CaM, but their roles at the physiological and molecular levels are largely unknown. We present data on Arabidopsis thaliana CML9 (AtCML9) that exhibits 46% amino acid sequence identity with CaM. AtCML9 transcripts are found in all major organs, and a putative AtCML9 regulatory region confers reporter gene expression at various sites, including root apex, stomata, hydathodes and trichomes. AtCML9 expression is rapidly induced by abiotic stress and abscisic acid (ABA) in young seedlings, and by using cml9 knock-out mutants we present evidence that AtCML9 plays essential roles in modulating responses to salt stress and ABA. Seed germination and seedling growth for the mutant lines present a hypersensitive response to ABA that could be correlated with enhanced tolerance to salt stress and water deficit. Mutations of the AtCML9 gene also alter the expression of several stress-regulated genes, suggesting that AtCML9 is involved in salt stress tolerance through its effects on the ABA-mediated pathways.
Summary 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.
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 2035 gene was expressed in Escherichiu 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 .Keywords: carboxylesterase ; serine esterase ; tobacco; plant; protein primary structure.Carboxylesterases constitute a ubiquitous class of enzymes, the members of which catalyze the hydrolysis of a broad range of compounds containing an ester linkage. They form a diverse family of enzymes that can be divided into several sub-groups (i.e. lipases, esterases) according to their specificity towards substrates. Lipases act almost exclusively on water-insoluble compounds such as triacylglycerides, and their activity is maximal when the enzyme is adsorbed onto a lipid water interface (Derewenda, 1994). The consequence of this property, known as the interfacial activation, is that most lipases are poor catalysts in a homogenous aqueous medium, where catalysis by esterases takes place. Although carboxylesterases hydrolyze vastly different substrates, substantial sequence similarity was recognized between members of this family. Despite a wide distribution throughout all living organisms, genes encoding lipases or esterases have been mainly isolated from mammals and microbes, and the comparison of numerous sequences revealed a significant similarity within the catalytic domain. A high degree of similarity was detected in a region surrounding a consensus sequence motif GXSXG, which contains the active site serine (Cygler et al., 1993 ;Derewenda and Sharp, 1993). Moreover, the three-dimensional struc-
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