Tzahi Arazi

Calmodulin binding to glutamate decarboxylase is required for regulation of glutamate and GABA metabolism and normal development in plants.

Glutamate decarboxylase (GAD) catalyzes the decarboxylation of glutamate to CO2 and gamma‐aminobutyrate (GABA). GAD is ubiquitous in prokaryotes and eukaryotes, but only plant GAD has been shown to bind calmodulin (CaM). Here, we assess the role of the GAD CaM‐binding domain in vivo. Transgenic tobacco plants expressing a mutant petunia GAD lacking the CaM‐binding domain (GADdeltaC plants) exhibit severe morphological abnormalities, such as short stems, in which cortex parenchyma cells fail to elongate, associated with extremely high GABA and low glutamate levels. The morphology of transgenic plants expressing the full‐length GAD (GAD plants) is indistinguishable from that of wild‐type (WT) plants. In WT and GAD plant extracts, GAD activity is inhibited by EGTA and by the CaM antagonist trifluoperazine, and is associated with a CaM‐containing protein complex of approximately 500 kDa. In contrast, GADdeltaC plants lack normal GAD complexes, and GAD activity in their extracts is not affected by EGTA and trifluoperazine. We conclude that CaM binding to GAD is essential for the regulation of GABA and glutamate metabolism, and that regulation of GAD activity is necessary for normal plant development. This study is the first to demonstrate an in vivo function for CaM binding to a target protein in plants.

Calcium/Calmodulin Activation of Soybean Glutamate Decarboxylase

Recently, we provided preliminary evidence for calcium (Ca2+)/calmodulin (CaM) stimulation of plant glutamate decarboxylase (GAD; EC 4.1.1.15). In the present study, a detailed characterization of the phenomenon is described. GAD was partially purified from various soybean (Glycine max L. Merr.) tissues (developing seed coat and cotyledons, leaf, and root) in the presence of EDTA by a combination of ammonium sulfate precipitation and anion-exchange fast protein liquid chromatography. GAD activity showed a sharp optimum at pH 5.8, with about 12% of maximal activity at pH 7. It was stimulated 2- to 8-fold (depending on the tissue source) in the presence of Ca2+/CaM at pH 7 but not at pH 5.8. Furthermore, when the protease inhibitor phenylmethylsulfonyl fluoride was omitted from the purification procedure, GAD activity was insensitive to Ca2+/CaM but was similar in magnitude to CaM-stimulated activity. The stimulation by Ca2+/CaM was fully inhibited by the CaM antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfon-amide and trifluoperazine. With saturating CaM or Ca2+, the concentrations of Ca2+ and CaM required for half-maximal stimulation were about 7 to 11 [mu]M and 25 nM, respectively. The effect of Ca2+ and CaM appeared to be through a 2.4-fold stimulation of Vmax and a 55% reduction in Km. The results suggested that GAD is activated via Ca2+ signal transduction.

Molecular and Biochemical Analysis of Calmodulin Interactions with the Calmodulin-Binding Domain of Plant Glutamate Decarboxylase

We previously provided what to our knowledge is the first evidence that plant glutamate decarboxylase (GAD) is a calmodulin (CaM)-binding protein. Here, we studied the GAD CaM-binding domain in detail. A synthetic peptide of 26 amino acids corresponding to this domain forms a stable complex with Ca2+/CaM with a 1:1 stoichiometry, and amino acid substitutions suggest that tryptophan-485 has an indispensable role in CaM binding. Chemical cross-linking revealed specific CaM/GAD interactions even in the absence of Ca2+. However, increasing KCl concentrations or deletion of two carboxy-terminal lysines abolished these interactions but had a mild effect on CaM/GAD interactions in the presence of Ca2+. We conclude that in the presence of Ca2+-hydrophobic interactions involving tryptophan-485 and electrostatic interactions involving the carboxy-terminal lysines mediate CaM/GAD complex formation. By contrast, in the absence of Ca2+, CaM/GAD interactions are essentially electrostatic and involve the carboxy-terminal lysines. In addition, a tryptophan residue and carboxy-terminal lysines are present in the CaM-binding domain of an Arabidopsis GAD. Finally, we demonstrate that petunia GAD activity is stimulated in vitro by Ca2+/CaM. Our study provides a molecular basis for Ca2+-dependent CaM/GAD interactions and suggests the possible occurrence of Ca2+-independent CaM/GAD interactions.

A high-affinity calmodulin-binding site in a tobacco plasma-membrane channel protein coincides with a characteristic element of cyclic nucleotide-binding domains

Recently we isolated a cDNA encoding a tobacco plasma membrane calmodulin-binding channel protein (designated NtCBP4) with a putative cyclic nucleotide-binding domain. Here we analyzed in detail the interaction of NtCBP4 with calmodulin. A full-length recombinant NtCBP4 (81 kDa) expressed in Sf9 insect cells, and the corresponding tobacco membrane protein were solubilized from their respective membrane fractions and partially purified by calmodulin affinity chromatography. NtCBP4 was detected in the eluted fractions using specific antibodies raised against the recombinant protein. By binding [35S]-calmodulin to recombinant NtCBP4 truncations fused to glutathione S-transferase, we identified a single region consisting of 66 amino acids capable of binding calmodulin. A 23 amino acid synthetic peptide from within this region formed a complex with calmodulin in the presence of calcium. We measured the fluorescence of dansyl-calmodulin interacting with this peptide, which revealed a dissociation constant of about 8 nM. The NtCBP4 calmodulin-binding domain was found to perfectly coincide with a phylogenetically conserved αC-helix motif of its putative cyclic nucleotide-binding domain. Furthermore, a 23 amino acid region in an equivalent site in the cAMP-binding domain of a mammalian protein kinase regulatory subunit was also found to bind calmodulin. Thus, coinciding calmodulin- and cyclic nucleotide-binding domains may serve as a point of communication between calcium and cyclic nucleotide signal transduction pathways in plants and animals.

Two isoforms of glutamate decarboxylase in Arabidopsis are regulated by calcium/calmodulin and differ in organ distribution

The nucleotide sequences of cDNAs encoding two isoforms of Arabidopsis glutamate decarboxylase, designated GAD1 (57.1 kDa) and GAD2 (56.1 kDa) and sharing 82% identical amino acid sequences, were determined. The recombinant proteins bound [35S] calmodulin (CaM) in the presence of calcium, and a region of 30–32 amino acids from the C-terminal of each isoform was sufficient for CaM binding when fused to glutathione S-transferase. Full-length GAD1 and GAD2 were expressed in Sf9 insect cells infected with recombinant baculovirus vectors. Recombinant proteins were partially purified by CaM affinity chromatography and were found to exhibit glutamate decarboxylase activity, which was dependent on the presence of Ca2+/CaM at pH 7.3. Southern hybridizations with GAD gene-specific probes suggest that Arabidopsis possesses one gene related to GAD1 and one to GAD2. Northern hybridization and western blot analysis revealed that GAD1 was expressed only in roots and GAD2 in roots, leaves, inflorescence stems and flowers. Our study provides the first evidence for the occurrence of multiple functional Ca2+/CaM-regulated GAD gene products in a single plant, suggesting that regulation of Arabidopsis GAD activity involves modulation of isoform-specific gene expression and stimulation of the catalytic activity of GAD by calcium signalling via CaM.

Cyclic-nucleotide- and Ca2+/calmodulin-regulated channels in plants: targets for manipulating heavy-metal tolerance, and possible physiological roles

Recently we discovered a tobacco protein (designated NtCBP4) that modulates heavy-metal tolerance in transgenic plants. Structurally, NtCBP4 is similar to mammalian cyclic-nucleotide-gated non-selective cation channels containing six putative transmembrane domains, a predicted pore region, a conserved cyclic-nucleotide-binding domain, and a high-affinity calmodulin-binding site that coincides with its cyclic-nucleotide-binding domain. Transgenic tobacco expressing the plasma-membrane-localized NtCBP4 exhibit improved tolerance to Ni(2+) and hypersensitivity to Pb(2+), which are associated with a decreased accumulation of Ni(2+) and an enhanced accumulation of Pb(2+) respectively. Transgenic plants expressing a truncated version of NtCBP4, from which regulatory domains had been removed, have a different phenotype. Here we describe our approach to studying the involvement of NtCBP4 in heavy-metal tolerance and to elucidate its physiological role.

Characterization of a Novel Family of Calmodulin-Binding Plasma Membrane Channel-Like Proteins

Calcium (Ca2+) is a ubiquitous second messenger in all eukaryotes (reviewed by Clapham, 1995; Bush, 1995; McAinsh and Hetherington, 1998). Ca2+ signals are transduced primarily by Ca2+ modulated proteins such as calmodulin (CaM) (Van Eldik and Watterson, 1998). CaM has no catalytic activity of its own and its diverse functions are the result of its interactions with numerous downstream effectors such as protein kinases, phosphatases, ion channels, and cytoskeleton associated proteins (James et al., 1995). The role of CaM and CaM-related proteins in plants is being unraveled in recent years (Snedden and Fromm, 1998; Zielinski, 1998). It became apparent that in spite of the high similarity of CaM from plants with that in animals (close to 90% identity in amino acid sequence) the downstream targets of CaM in plants and animals are not all the same (Baum et al., 1996; Snedden and Fromm, 1998). In addition, plants possess a large repertoire of calmodulin-related proteins not present in other eukaryotes (Snedden and Fromm, 1998). Thus, plants use the Ca2+/CaM messenger system in unique ways to accommodate their physiological requirements.

Redistribution of CHH Methylation and Small Interfering RNAs across the Genome of Tomato ddm1 Mutants

The production of siRNAs and the CHH methylation mediated by the RdDM pathway are enhanced in heterochromatin when DDM1 is nonfunctional, at the expense of silencing mechanisms occurring in euchromatin. In plants, cytosine methylation, an epigenetic mark critical for transposon silencing, is maintained over generations by key enzymes that directly methylate DNA and is facilitated by chromatin remodelers, like DECREASE IN DNA METHYLATION1 (DDM1). Short-interfering RNAs (siRNAs) also mediate transposon DNA methylation through a process called RNA-directed DNA methylation (RdDM). In tomato (Solanum lycopersicum), siRNAs are primarily mapped to gene-rich chromosome arms, and not to pericentromeric regions as in Arabidopsis thaliana. Tomato encodes two DDM1 genes. To better understand their functions and interaction with the RdDM pathway, we targeted the corresponding genes via the CRISPR/Cas9 technology, resulting in the isolation of Slddm1a and Slddm1b knockout mutants. Unlike the single mutants, Slddm1a Slddm1b double mutant plants display pleiotropic vegetative and reproductive phenotypes, associated with severe hypomethylation of the heterochromatic transposons in both the CG and CHG methylation contexts. The methylation in the CHH context increased for some heterochromatic transposons and conversely decreased for others localized in euchromatin. We found that the number of heterochromatin-associated siRNAs, including RdDM-specific small RNAs, increased significantly, likely limiting the transcriptional reactivation of transposons in Slddm1a Slddm1b. Taken together, we propose that the global production of siRNAs and the CHH methylation mediated by the RdDM pathway are restricted to chromosome arms in tomato. Our data suggest that both pathways are greatly enhanced in heterochromatin when DDM1 functions are lost, at the expense of silencing mechanisms normally occurring in euchromatin.

Calmodulin and Plant Responses to the Environment

Calcium ions (Ca2+) function as a widespread messenger in mediating responses of eukaryotes to environmental signals, often through Ca2+-modulated proteins like calmodulin (CaM) (for review see Snedden and Fromm 1998, Zielinski 1998). To understand how plants respond to environmental changes we focus on the identification and characterisation of the cellular targets of Ca2+/CaM. These proteins are believed to play a key role in regulating cellular and biochemical processes associated with plant adaption to the environment (e.g. metabolic regulation, transcriptional regulation, ion transport and cell structure modifications).