Ca2+ and peroxidase derived from cultured peanut cells

A 5% increase of Ca²⁺ content of the incubation medium for cultured peanut ( Arachis hypogaea L.) cells caused a rise of peroxidase (EC 1.11.1.7) activity in the medium, which could be abolished by the addition of the chelator EGTA [eth-yleneglycol-bis-(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid]. However, the determination of in vivo peroxidase synthesis in these cells showed that Ca²⁺ had a direct effect on the biosynthesis rather than on transport alone. This concept was re-enforced by the lack of effect by the ionophore A23187 on the transport. The Ca²⁺ content was 2 and 5 mol (mol protein⁻¹) for the cationic and anionic peanut peroxidase, respectively. The latter is different from the value reported for the anionic peroxidase from horseradish.     

Recognition between tRNASer and archaeal seryl-tRNA synthetases monitored by suppression of bacterial amber mutations

Two dissimilar seryl-tRNA synthetases (SerRSs) exist in Methanosarcina barkeri : one of bacterial type (bMbSerRS) and the other resembling SerRSs present only in methanogenic archaea (mMbSerRS). While the expression of the archaeal bMbSerRS gene in Escherichia coli complements the function of thermolabile SerRS at a nonpermissive temperature, mMbSerRS does not. Our recent X-ray structural analysis of mMbSerRS revealed an idiosyncratic N-terminal domain and a catalytic zinc ion in the active site, identifying methanogenic-type SerRSs as atypical members of the SerRS family. To shed further light on substrate discrimination by methanogenic-type SerRS, we developed an in vivo system in E. coli to study tRNA serylation by mMbSerRS variants. We show that coexpression of the M. barkeri SerRS gene, encoding either bacterial- or methanogenic-type SerRS, with the gene for cognate archaeal suppressor tRNA leads to suppression of bacterial amber mutations, implying that the E. coli translation machinery can use serylated tRNA from methanogenic archaea as a substrate in protein synthesis. Furthermore, because serylation of M. barkeri serine-specific tRNA by endogenous E. coli SerRS is negligible, suppression is entirely dependent on recognition between archaeal partners (mMbSerRS/suppressor tRNA^Ser). Thus, the efficiency of suppression by mMbSerRS variants quantified in the described β-galactosidase-based reporter system, accurately reflects enzymes' serylation propensity obtained by in vitro kinetic measurements.     

Kinetics of efflux of K+ from cultured rose cells treated with ultraviolet light

Irradiation of cultured rose ( Rosa damascena Mill. cv. Gloire de Guilan) cells with ultraviolet light caused of loss of K⁺, which occurred with sigmoid kinetics. The kinetics of loss of K⁺ were not changed when the extracellular concentration of K⁺ was held constant during the period of efflux. Furthermore, the rate of loss of K⁺ was approximately the same even though the K⁺ concentration in the medium was increased from 0.1 to 10 m M . The kinetics of uptake of the lipophilic methyltriphenylphosphonium cation, an indicator of the plasma membrane potential, were linear throughout the period of K⁺ efflux, suggesting that the starting and stopping of K⁺ efflux do not reflect a passive response to changes in the membrane potential of the cells. The results are interpreted in terms of activation and inactivation of an efflux channel or pump for K⁺.     

In vitro analysis of a type I DNA topoisomerase activity from cultured tobacco cells

The role of DNA topoisomerases in plant cell metabolism is currently under investigation in our laboratory. Using a purified type I topoisomerase from cultured tobacco, we have carried out a biochemical characterization of enzymatic behavior. The enzyme relaxes negatively supercoiled DNA in the presence of MgCl₂, and to a lesser extent in the presence of KCl. Phosphorylation of the topoisomerase does not influence its activity and it is not stimulated by the presence of histones H1 or H5. The enzyme may act in either a processive or distributive manner depending on reaction conditions. The anti-tumor drug, camptothecin, induces significant breakage by the enzyme on purified DNA molecules unless destabilized by the addition of KCl. The tobacco topoisomerase I can catalyze the formation of stable nucleosomes on circular DNA templates, suggesting a role for the enzyme in chromatin assembly.     

In vitro processing of a plant pre-mRNA in a HeLa cell nuclear extract.

In order to determine whether there is a general difference in the splicing mechanism of animal and plant pre-mRNAs, we cloned part of the gene for the small subunit of the ribulose 1,5-bisphosphate carboxylase containing both introns into the SP64 vector. RNA was synthesized with SP6 polymerase and used as substrate for in vitro processing in a HeLa cell nuclear splicing extract. Analyses of the processed RNA demonstrate that both introns of the plant pre-mRNA are efficiently removed in an ordered fashion yielding a faithfully ligated mRNA. Two branch points were identified for intron A and three for intron B. The branched nucleotides are adenosine residues in all cases and are located within a distance from the 3' splice site found to be crucial for lariat formation in animal pre-mRNAs. The implications of these results are discussed in light of our previous observation, that a functional pre-mRNA of the human growth hormone gene was not processed in plant tissue in vivo.     

Ca2+ dependence and La3+ interference of ultraviolet radiationinduced K+ efflux from rose cells

A low fluence of ultraviolet radiation (UV) causes cultured cells of Rosa damascena Mill cv. Gloire de Guilan to lose intracellular K⁺. This effect required the presence of Ca²⁺ in the medium. A reduction in the concentration of free Ca²⁺ to 10⁻⁵ M with ethyleneglycol-bis-(β-aminoethyl-ether)-N.N.N',N'-tetraacetic acid (EGTA) buffer inhibited the UV-stimulated efflux; this was correlated with a discharge of the membrane potential and a stimulation of the leakage of K⁺ from unirradiated cells. All the same effects were seen with La³⁺ at 0.2 m M. At 0.02 m M La³⁺, the UV-stimulated efflux of K⁺ was blocked without concomitant effects on the membrane potential or K⁺ efflux from control cells. It is suggested that removal of Ca²⁺ blocks or masks the UV-induced leakage of K⁺ by destabilizing the plasma membrane. In addition, La³⁺ may specifically inhibit the UV-stimulated opening of K⁺ or anion channels.     

Effect of auxin on alkaloids, K+ and free amino acid content in cultured tobacco callus

Callus cultures derived from the petiole of Nicotiana tabacum L. cv. Burley 21 were grown at 25°C in the dark on two different basal media containing: (1) 11.5 μ M α-naphthaleneacetic acid and 1 μ M kinetin, and (2) 1 μ M α-naphthaleneacetic acid and 1 μ M kinetin. The contents of alkaloids, K⁺ and free amino acids of callus tissues were determined. The tissues were also examined microscopically for organization when organogenesis was not apparent. The first medium limited nicotine synthesis and stimulated its N-demethylation to nornicotine. The second medium stimulated nicotine synthesis and limited tissue growth. Although significantly higher concentrations of K⁺ were observed in calli grown on the high-auxin medium, both cultures were K⁺ deficient. The fact that the low-auxin medium limited K⁺ uptake to a higher degree would account for the lower growth observed in calli cultured on this medium, and it is possible that the effect of auxin concentration on nicotine production may be mediated through its effects on K⁺ uptake by cells of the culture. The free amino acid concentration increased in the calli grown on the low-auxin medium. Glutamic acid and proline, known as initial precursors of nicotine, increased significantly. Histological examination showed that the occurrence of meristematic areas in calli without organogenesis promoted nicotine synthesis. The relation between the accumulation of nicotine and formation of roots or shoot-buds is discussed.     

Vitamin D3 affects growth and Ca2+ uptake by Phaseolus vulgaris roots cultured in vitro

Vitamin D₃ at low concentration (10⁻⁹ M) inhibited the growth of Phaseolus vulgaris L. (cv. Contrancha) roots in vitro as measured by elongation (14 h) and [³H]-leucine incorporation into protein (2 h), and increased their labelling with ⁴⁵Ca²⁺ (2 h). Cycloheximide and puromycin (50 u.M) blocked vitamin D₃ stimulation of root ⁴⁵Ca²⁺ labelling, indicating that it is mediated by de novo protein synthesis. The calcium ionophore X-537A (10⁻⁵JW) induced similar changes both in root elongation and ⁴⁵Ca²⁺ uptake (14 h). This may indicate that the inhibitory effects of the sterol on root growth are mediated by changes in Ca²⁺ fluxes. However, this interpretation should be further strengthened by additional studies as the ionophore may have acted on root growth, affecting physiological processes other than Ca²⁺ transport.     

Control of phosphatase release from cultured tobacco cells

The activity of cultured tobacco XD-6 cells to release phosphataseinto the medium was enhanced after a time lag of 2 to 3 daysfollowing a marked increase in intracellular level of the enzymeduring Pi-omitted culture. The enhanced release of phosphatasehad a Q₁₀ value of about 2.3, and was suppressed by 2,4-dinitrophenoland azide. Cycloheximide did not inhibit the enzyme release,but Pi caused a rapid and drastic decrease in the rate of release.The release is suppressed even by 1 µM Pi. These results indicate that the rate of enzyme release is notdirectly limited by synthesis of the enzyme, but limited bythe transport, which may be suppressed by Pi, of the enzymeto the outside of the cell membrane. ¹ Present address: Laboratory of Applied Microbiology, Facultyof Agriculture, Yamagata University, Tsuruoka, Yamagata-ken997, Japan. (Received June 28, 1977; )