Isolation and characterization of a novel calmodulin-binding protein from potato.

Tuberization in potato is controlled by hormonal and environmental signals. Ca(2+), an important intracellular messenger, and calmodulin (CaM), one of the primary Ca(2+) sensors, have been implicated in controlling diverse cellular processes in plants including tuberization. The regulation of cellular processes by CaM involves its interaction with other proteins. To understand the role of Ca(2+)/CaM in tuberization, we have screened an expression library prepared from developing tubers with biotinylated CaM. This screening resulted in isolation of a cDNA encoding a novel CaM-binding protein (potato calmodulin-binding protein (PCBP)). Ca(2+)-dependent binding of the cDNA-encoded protein to CaM is confirmed by (35)S-labeled CaM. The full-length cDNA is 5 kb long and encodes a protein of 1309 amino acids. The deduced amino acid sequence showed significant similarity with a hypothetical protein from another plant, Arabidopsis. However, no homologs of PCBP are found in nonplant systems, suggesting that it is likely to be specific to plants. Using truncated versions of the protein and a synthetic peptide in CaM binding assays we mapped the CaM-binding region to a 20-amino acid stretch (residues 1216-1237). The bacterially expressed protein containing the CaM-binding domain interacted with three CaM isoforms (CaM2, CaM4, and CaM6). PCBP is encoded by a single gene and is expressed differentially in the tissues tested. The expression of CaM, PCBP, and another CaM-binding protein is similar in different tissues and organs. The predicted protein contained seven putative nuclear localization signals and several strong PEST motifs. Fusion of the N-terminal region of the protein containing six of the seven nuclear localization signals to the reporter gene beta-glucuronidase targeted the reporter gene to the nucleus, suggesting a nuclear role for PCBP.

Temperature-jump relaxation kinetics of substrate-induced spin-state transition in cytochrome P450 (comparison of the wild-type and C334A mutant P450(CAM) and P450(2B4)).

The kinetics of binding of the substrate camphor to the cytochrome P450(CAM) and the C334A mutant as well as the kinetics of binding of benzphetamine to the wild-type P450(2B4) have been studied by the temperature-jump relaxation technique in order to distinguish between the two models for substrate-induced spin-state transition. These models are the bimolecular model in which spin-state transition occurs in parallel with substrate binding, and the two-step spin-equilibrium model in which substrate binding is a separate step preceding the spin-state transition. With all three P450s, the relaxation rate versus concentration data were linear as predicted by the bimolecular model and inconsistent with the spin-equilibrium model, which predicts a curve reaching saturation. With all three P450s, the relaxation rate versus concentration data exhibited maxima. These results are considered to resolve the controversy in favor of the bimolecular model for substrate-induced spin-state transition. In addition, the results suggest that the bimolecular model may be applicable to other P450s as well.

Characterization of microtubule binding domains in the Arabidopsis kinesin-like calmodulin binding protein.

The kinesin-like calmodulin binding protein (KCBP) is a new member of the kinesin superfamily that appears to be present only in plants. The KCBP is unique in its ability to interact with calmodulin in a Ca2+-dependent manner. To study the interaction of the KCBP with microtubules, we expressed different regions of the Arabidopsis KCBP and used the purified proteins in cosedimentation assays with microtubules. The motor domain with or without the calmodulin binding domain bound to microtubules. The binding of the motor domain containing the calmodulin binding region to microtubules was inhibited by Ca2+-calmodulin. This Ca2+-calmodulin regulation of motor domain interactions with microtubules was abolished in the presence of antibodies specific to the calmodulin binding region. In addition, the binding of the motor domain lacking the calmodulin binding region to microtubules was not inhibited in the presence of Ca2+-calmodulin, suggesting an essential role for the calmodulin binding region in Ca2+-calmodulin modulation. Results of the cosedimentation assays with the N-terminal tail suggest the presence of a second microtubule binding site on the KCBP. However, the interaction of the N-terminal tail region of the KCBP with microtubules was insensitive to ATP. These data on the interaction of the KCBP with microtubules provide new insights into the functioning of the KCBP in plants.

Interactions of substrate and product with cytochrome P450: P4502B4 versus P450cam.

In the present study, two P450s (P4502B4 and P450cam) have been examined with regard to their interactions with their substrates and products utilizing the characteristic spectral perturbations as criteria for their binding. The results indicate that although there are differences between the two P450s (E) in regard to their precise interactions with their substrates (S) and products (P), the spectral titration data were consistent with the two-site model--E + S<-->ES (K1), E + P<-->EP (K2); EP + S<-->ESP (K3); ES + P<-->ESP (K4) in which S and P bind to E forming ESP. The data were inconsistent with the two-site model in which S and P compete for the same site. As required by the two-site model, the relationship K2K3 = K1K4 was maintained with both P450s at all product concentrations tested, although K3 and K4 decreased considerably when product concentration was increased. The relationship K3 >> K4 was also maintained, indicating that with both enzymes' ESP is formed predominantly by binding of S to EP rather than binding of P to ES, and that ESP dissociates predominantly to ES and P rather than EP and S. In other words, binding of S to EP facilitates the dissociation of P. This indicates that the relative parameter values are compatible for ESP to have functional significance. The possible role of ESP in controlling catalytic rate and catalytic efficiency is discussed.

Interaction of Arabidopsis kinesin-like calmodulin-binding protein with tubulin subunits: modulation by Ca(2+)-calmodulin.

Kinesin-like calmodulin-binding protein (KCBP) is a recently identified novel kinesin-like protein that appears to be unique to and ubiquitous in plants. KCBP is distinct from all other known KLPs in having a calmodulin-binding domain adjacent to its motor domain. We have used different regions of KCBP to study its interaction with tubulin subunits and the regulation of this interaction by Ca(2+)-calmodulin. The results show that the carboxy-terminal part of the KCBP, with or without calmodulin-binding domain, binds to tubulin subunits and this binding is sensitive to nucleotides. In the presence of Ca(2+)-calmodulin the motor with calmodulin-binding domain does not bind to tubulin. This Ca(2+)-calmodulin modulation is abolished in the presence of antibodies specific to the calmodulin-binding domain of KCBP. Similar binding studies with the carboxy-terminal part of KCBP lacking the calmodulin-binding domain show no effect of Ca(2+)-calmodulin. These results indicate that Ca(2+)-calmodulin modulates the interaction of KCBP with tubulin subunits and this modulation is due to the calmodulin-binding domain in the KCBP. Calcium-dependent calmodulin modulation of KCBP interaction with tubulin suggests regulation of KCBP function by calcium, the first such regulation of a kinesin heavy chain among all the known kinesin-like proteins.

Structural organization of a gene encoding a novel calmodulin-binding kinesin-like protein from Arabidopsis.

Kinesin-like calmodulin-binding protein (KCBP) is a recently identified microtubule motor protein that appears to be unique to plants. Here we report isolation and sequence analysis of a gene encoding Arabidopsis KCBP. KCBP gene contains 21 exons and 20 introns. All exons except exon 3 are short (94-272 nt). Exons 1-9 code for the globular tail region whereas the coiled-coil region is coded by exons 10-15. The conserved motor domain is coded by exons 16-20. Calmodulin-binding domain that is present in the C-terminal region of the protein and unique to KCBP is coded by the last exon. The size of introns ranged from 71 (intron 17) to 320 (intron 19) nucleotides. As in most plant introns, the content of AT is very high in all introns (up to 76%). Phylogenetic analysis of KCBP using motor domain sequence grouped KCBP with other known C-terminal microtubule motor proteins. However, Arabidopsis KCBP together with its homologs from potato and tobacco constitute a distinct group within the C-terminal subfamily of motors which is consistent with structural and functional features of KCBP.

A plant kinesin heavy chain-like protein is a calmodulin-binding protein.

Calmodulin, a calcium modulated protein, regulates the activity of several proteins that control cellular functions. A cDNA encoding a unique calmodulin-binding protein, PKCBP, was isolated from a potato expression library using protein-protein interaction based screening. The cDNA encoded protein bound to biotinylated calmodulin and 35S-labeled calmodulin in the presence of calcium and failed to bind in the presence of EGTA, a calcium chelator. The deduced amino acid sequence of the PKCBP has a domain of about 340 amino acids in the C-terminus that showed significant sequence similarity with the kinesin heavy chain motor domain and contained conserved ATP- and microtubule-binding sites present in the motor domain of all known kinesin heavy chains. Outside the motor domain, the PKCBP showed no sequence similarity with any of the known kinesins, but contained a globular domain in the N-terminus and a putative coiled-coil region in the middle. The calmodulin-binding region was mapped to a stretch of 64 amino acid residues in the C-terminus region of the protein. The gene is differentially expressed with the highest expression in apical buds. A homolog of PKCBP from Arabidopsis (AKCBP) showed identical structural organization indicating that kinesin heavy chains that bind to calmodulin are likely to exist in other plants. This paper presents evidence that the motor domain has microtubule stimulated ATPase activity and binds to microtubules in a nucleotide-dependent manner. The kinesin heavy chain-like calmodulin-binding protein is a new member of the kinesin superfamily as none of the known kinesin heavy chains contain a calmodulin-binding domain. The presence of a calmodulin-binding motif and a motor domain in a single polypeptide suggests regulation of kinesin heavy chain driven motor function(s) by calcium and calmodulin.

Early transcription of Agrobacterium T-DNA genes in tobacco and maize.

We developed a sensitive procedure to investigate the kinetics of transcription of an Agrobacterium tumefaciens transferred (T)-DNA-encoded beta-glucuronidase gusA (uidA) gene soon after infection of plant suspension culture cells. The procedure uses a reverse transcriptase-polymerase chain reaction and enables detection of gusA transcripts within 18 to 24 hr after cocultivation of the bacteria with either tobacco or maize cells. Detection of gusA transcripts depended absolutely on the intact virulence (vir) genes virB, virD1/virD2, and virD4 within the bacterium. Mutations in virC and virE resulted in delayed and highly attenuated expression of the gusA gene. A nonpolar transposon insertion into the C-terminal coding region of virD2 resulted in only slightly decreased production of gusA mRNA, although this insertion resulted in the loss of the nuclear localization sequence and the important omega region from VirD2 protein and rendered the bacterium avirulent. However, expression of gusA transcripts in tobacco infected by this virD2 mutant was more transient than in cells infected by a wild-type strain. Infection of tobacco cells with an Agrobacterium strain harboring a mutant virD2 allele from which the omega region had been deleted resulted in similar transient expression of gusA mRNA. These data indicate that the C-terminal nuclear localization signal of the VirD2 protein is not essential for nuclear uptake of T-DNA and further suggest that the omega domain of VirD2 may be required for efficient integration of T-DNA into the plant genome. The finding that the initial kinetics of gusA gene expression in maize cells are similar to those shown in infected tobacco cells but that the presence of gusA mRNA in maize is highly transient suggests that the block to maize transformation involves T-DNA integration and not T-DNA entry into the cell or nuclear targeting.

A novel kinesin-like protein with a calmodulin-binding domain.

Calcium regulates diverse developmental processes in plants through the action of calmodulin. A cDNA expression library from developing anthers of tobacco was screened with 35S-labeled calmodulin to isolate cDNAs encoding calmodulin-binding proteins. Among several clones isolated, a kinesin-like gene (TCK1) that encodes a calmodulin-binding kinesin-like protein was obtained. The TCK1 cDNA encodes a protein with 1265 amino acid residues. Its structural features are very similar to those of known kinesin heavy chains and kinesin-like proteins from plants and animals, with one distinct exception. Unlike other known kinesin-like proteins, TCK1 contains a calmodulin-binding domain which distinguishes it from all other known kinesin genes. Escherichia coli-expressed TCK1 binds calmodulin in a Ca(2+)-dependent manner. In addition to the presence of a calmodulin-binding domain at the carboxyl terminal, it also has a leucine zipper motif in the stalk region. The amino acid sequence at the carboxyl terminal of TCK1 has striking homology with the mechanochemical motor domain of kinesins. The motor domain has ATPase activity that is stimulated by microtubules. Southern blot analysis revealed that TCK1 is coded by a single gene. Expression studies indicated that TCK1 is expressed in all of the tissues tested. Its expression is highest in the stigma and anther, especially during the early stages of anther development. Our results suggest that Ca2+/calmodulin may play an important role in the function of this microtubule-associated motor protein and may be involved in the regulation of microtubule-based intracellular transport.

A novel plant calmodulin-binding protein with a kinesin heavy chain motor domain.

Calmodulin, a ubiquitous calcium-binding protein, regulates many diverse cellular functions by modulating the activity of the proteins that interact with it. Here, we report isolation of a cDNA encoding a novel kinesin-like calmodulin-binding protein (KCBP) from Arabidopsis using biotinylated calmodulin as a probe. Calcium-dependent binding of the cDNA-encoded protein to calmodulin is confirmed by 35S-labeled calmodulin. Sequence analysis of a full-length cDNA indicates that it codes for a protein of 1261 amino acids. The predicted amino acid sequence of the KCBP has a domain of about 340 amino acids in the COOH terminus that shows significant sequence similarity with the motor domain of kinesin heavy chains and kinesin-like proteins and contains ATP and microtubule binding sites typical of these proteins. Outside the motor domain, the KCBP has no sequence similarity with any of the known kinesins, but contains a globular domain in the NH2 terminus and a putative coiled-coil region in the middle. By analyzing the calmodulin binding activity of truncated proteins expressed in Escherichia coli, the calmodulin binding region is mapped to a stretch of about 50 amino acid residues in the COOH terminus region of the protein. Using a synthetic peptide, the calmodulin binding domain is further narrowed down to a 23-amino acid stretch. The synthetic peptide binds to calmodulin with high affinity in a calcium-dependent manner as judged by electrophoretic mobility shift assay of calmodulin-peptide complex. The KCBP is coded by a single gene and is highly expressed in developing flowers and suspension cultured cells. Although many kinesin heavy chains and kinesin-like proteins have been extensively characterized at the biochemical and molecular level in evolutionarily distant organisms, none of them is known to bind calmodulin. The plant kinesin-like protein with a calmodulin binding domain and a unique amino-terminal region is a new member of the kinesin superfamily. The presence of a calmodulin-binding motif in a kinesin heavy chain-like protein suggests a role for calcium and calmodulin in kinesin-driven motor function(s) in plants.

Interactions of substrate and product with cytochrome P450 2B4.

Interactions of the substrate(s) benzphetamine and the product (P) desmethylbenzphetamine with cytochrome P450 2B4 were studied by difference spectrophotometry. A two-sites model in which site 1 binding, causing Type I transition (low- to high-spin) must precede site 2 binding, causing Type II transition (high- to low-spin), gave an acceptable fit to the spectral titration data. The equilibrium association constant of substrate for site 1 (K1) was greater than that for site 2 (K2), and the K2 for the product was greater than K1, indicating that the substrate binds preferentially to site 1 and the product prefers site 2. In addition, competition between P and a strong Type II ligand (1-benzylimidazole) and a noncompetitive type of interaction between S and the same Type II ligand was observed. This indicates that P binds to the same site as the Type II ligand and S binds to a different site. The observed high-spin maxima for both P (EP1HSmax) and S (ES1HSmax) were similar to those calculated using the K1 and K2 values obtained from the curve-fitting procedure, indicating that the equilibrium concentration of the high-spin species is controlled entirely by K1 and K2. Simultaneous presence of the substrate and product decreased K1 of the substrate and K2 of the product, indicating that there is interaction between the substrate-preferred and the product-preferred sites. A possible functional significance of the differences in the site preferences of the substrate and product is discussed.

Intergeneric protoplast fusion between Brassica carinata and Camelina sativa.

Camelina sativa is a wild crucifer that is reported to be resistant to Alternaria blight. Polyethylene glycol mediated fusion was attempted between protoplasts from etiolated hypocotyls of Brassica carinata and mesophyll protoplasts of Camelina sativa. The mean frequency of heterokaryons was 6.8%. Three hybrid shoots were regenerated, each from a single fusionderived callus. These shoots failed to produce roots capable of withstanding transplantation. Confirmation of hybridity was obtained from the morphology of in vitro produced leaves, somatic chromosome number in leaf tips, and restriction fragment length polymorphism for a nuclear rDNA probe. Analysis for organelle constitution using RFLPs indicated that the hybrid contained chrloroplasts derived from the wild species and mitochondria from the cultivated Brassica species.

On the model controversy for substrate-induced spin-state transition in cytochrome P450: (a new perspective).

Three models have been proposed for substrate-induced spin-state transition in cytochrome P450. These are referred to as two-, three- and four-state models. In this communication the three models are reviewed with respect to their experimental basis and their ability to accommodate the results reported on the effects of substrates on spin-state and reduction of various P450's. In addition, a new perspective is presented.

Substrate-induced spin-state transition in cytochrome P450LM2: a temperature-jump relaxation study.

The kinetics of the benzphetamine-P450LM2 binding reaction were studied by the T-jump relaxation technique, using the substrate-induced type I spectral change (which reflects transformation of the heme from the low- to the high-spin state) as the criterion for binding. The reciprocal relaxation time (kobs) exhibited a linear dependence on [E]eq+[S]eq. The kinetically determined dissociation equilibrium constant (68 +/- 10 microM) and that determined by direct titration of the spectral change (61 +/- 4 microM) were very similar. These results indicate that the substrate-induced spin-state transition follows a simple biomolecular binding mechanism; that is, the substrate-induced low- to high-spin transition reflects substrate binding.

Shoot regeneration in stem expiants and its amenability to Agrobacterium tumefaciens mediated gene transfer in Brassica carinata.

Immature stem segments of seven different genotypes of Brassica carinata produced shoots with variable frequencies when cultured in MS medium with BAP and picloram at 0.2 mg/l each. Line 171, which produced shoots with 100% efficiency from both cut ends of the expiant, was selected for testing the amenability of this regeneration protocol for genetic transformation. A non-oncogenic Agrobacterium tumefaciens containing plasmid PCV 730, a binary vector carrying resistance genes for kanamycin and hygromycin, was used. A cocultivation period of 4 d with a bacterial concentration of approximately 2.5×10 cells/ml, followed by a recovery period of 2 d, produced transformed shoots that could be selected and rooted in the presence of kanamycin at 15 mg/l. Transformation was confirmed by neomycin phospho-transferase assay and Southern blot analysis. Seed analysis of transformed plants indicated that kanamycin resistance was inherited in the progeny.

Resynthesis of Brassica carinata by protoplast fusion and recovery of a novel cytoplasmic hybrid.

Brassica carinata (2n=34, BBCC), was synthesized by fusing dark grown etiolated hypocotyl protoplasts of B. nigra (2n=16, BB) with green mesophyll protoplasts of B. oleracea (2n=18,CC) using polyethylene glycol. Heterokaryons could be microscopically distinguished from the parental types by their dark green chloroplasts in the colourless hypocotyl protoplast background. The mean heterokaryotic fusion frequency estimated on the basis of this morphological distinction was about 16%. A total of 626 calli were obtained, of which 92 regenerated shoots after transfer to zeatin (2 mg/l) supplemented MS medium. Of these, 81 calli differentiated into plants morphologically similar to naturally occurring B. carinata and 11 calli yielded plants resembling parental types. Meiosis in seven hybrid plants showed the chromosome number to be 2n=34 the sum of B. nigra and B. oleracea chromosomes. Molecular confirmation of the amphidiploid nature of hybrids was obtained by probing with a B. juncea derived genomic clone. The use of chloroplast and mitochondrial specific gene probes, revealed that one plant was a cytoplasmic hybrid having cp DNA sequences of both B. oleracea and B. nigra and mt DNA sequences of B. nigra.

Somatic hybridization between Brassica juncea and Moricandia arvensis by protoplast fusion.

Intergeneric somatic hybrids have been produced between Brassica juncea (2n=36, AABB) cv. RLM-198 and Moricandia arvensis (2n=28, MM) by protoplast fusion. Hypocotyl protoplasts of B. juncea were fused with mesophyll protoplasts of M. arvensis using polyethylene glycol. Fusion frequency, estimated on the basis of differential morphological characterstics of parental protoplasts was about 5%. Of the 156 calli obtained, four calli produced shoots intermediate in morphology between the parents. Hybrid nature of the plants was confirmed using wheat nuclear rDNA probe. Hybridization of total DNA with a mitochondrial DNA probe carrying 5s-18s rRNA genes of maize showed that the mitochondria of the somatic hybrids were derived from the wild species M. arvensis. Meiosis in the only hybrid that produced normal flowers revealed the occurrence of 64 chromosomes, the sum of chromosomes of parental species. Inspite of complete pollen sterility, siliquas were produced in this hybrid by back-crossing with B. juncea. These siliquas on in vitro culture produced 12 seeds.

Production and characterization of intergeneric somatic hybrids of Trachystoma ballii and Brassica juncea.

Intergeneric somatic hybrids, Trachystoma ballii (2n=16)+B. juncea (2n=38), were obtained by fusing mesophyll protoplasts of T. ballii and hypocotyl protoplasts of B. juncea using polyethylene glycol. The heterokaryotic fusion frequency was around 23%. Plants were regenerated from 10 out of 2×10(4) calli of which four were hybrids. Hybrids were intermediate between the parents in general morphology. However, in some characters one of the parents dominated. All the plants were symmetric in their chromosomal constitution as revealed by the formation of 26 bivalents at metaphase-I of meiosis. Two trivalents and 2 univalents were also observed in some pollen mother cells. Hybrid nature was also confirmed by 'Southern' hybridization of DNA of one regenerated plant restricted with Hind III and probed with the nick translated plasmid pTA71 carrying a wheat nuclear r-DNA sequence. Hybrid plant RT 1 showed bands characteristic of both parents. All the plants were absolutely pollen sterile. However, on backcrosses to B. juncea seeds were obtained.

Rapid and efficient plant regeneration from hypocotyl protoplasts of Brassica carinata.

Protoplasts were isolated from hypocotyls of 7-d-old seedlings of three genotypes of Brassica carinata after enzymatic digestion in cellulase R-10 (0.5%) and pectolyase Y-23 (0.025%). The protoplasts were stabilized with 0.4 M mannitol used as osmoticum, and were cultured in darkness in Kao's liquid medium containing 0.4 M glucose and the growth regulators 2,4-D (1.0 mg/l), NAA (0.1 mg/l) and zeatin riboside (0.5 mg/l). Protoplasts were transferred to 16 h photoperiod conditions after 3 d of dark culture, and the medium was diluted to reduce the osmoticum on the seventh and tenth days of culture. Microcolonies were thus obtained which, upon transfer to MS agarose medium with 2,4-D (0.1 mg/l), BAP (1 mg/l) and 0.1 M sucrose, proliferated further to produce callus clumps. The plating efficiency of the three genotypes varied from 1 to 2%. Calli 2-3 mm in diameter were transferred to MS agarose plates with zeatin (2 mg/l) where they produced shoot buds and shoots with frequencies ranging from 22.5 to 74.2% for the three genotypes. The shoots were rooted in medium with IBA (1 mg/l) and were then established in soil. The time required for protoplast to plant development was 8 to 10 weeks.