The diffusive transport of gibberellins and abscisic acid through the aleurone layer of germinating barley grain: a mathematical model.

A mathematical model of the diffusive transport of abscisic acid (ABA) and gibberellins (GAs) through the aleurone layer of barley (Hordeum vulgare L.) grain is presented. The model consists of two partial differential equations describing the accumulation of phytohormone in the apoplastic and symplasmic compartments of the aleurone layer, both spatially and temporally. The mathematical model contains the morphology of the barley grain and the physicochemical properties of the two phytohormones. A mathematical derivation of the accumulation ratios for the two phytohormones between the symplast and apoplast under equilibrium conditions resulted in different distribution mechanisms for GAs and ABA. A sensitivity analysis of the accumulation ratio for GAs indicated high sensitivity to the apoplastic pH and the membrane potential, whereas the accumulation ratio for ABA proved to be most sensitive to the pH difference between the apoplast and symplast. The diffusive transport time for GAs to the basal site of the aleurone layer as calculated with the mathematical model is within a physiologically plausible timescale according to experimental data from the literature. Abscisic acid cannot be transported by diffusion to the end of the aleurone layer as quickly as GAs, according to model simulations. Therefore, the functional role of ABA in germination is likely to be in the vicinity of the embryo.

Hexose uptake by Catharanthus roseus cell suspensions is inhibited by a high medium salt content.

The uptake of glucose and fructose from the medium by Catharanthus roseus cell suspensions was strongly inhibited by high medium salt concentration, such as found in LS (Linsmaier and Skoog 1965) medium. After inoculation into standard LS nutrient medium with less than 5 mM hexose no uptake occurred, while in low salt medium hexose was completely depleted. At a hexose concentration of 50 mM the uptake rate was higher in low salt medium than in standard medium. The lower rate of uptake at high salt concentration was not the result of a pH or osmotic effect of the salts. Probably the affinity of the hexose carrier is affected by the ion concentration of the medium. The decrease in medium salt concentration during normal batch culture probably will have a considerable effect on hexose uptake.

Further Characterization of Expression of Auxin-Induced Genes in Tobacco (Nicotiana tabacum) Cell-Suspension Cultures.

We have described the modulation of four auxin-regulated genes during the growth cycle of suspension-cultured tobacco (Nicotiana tabacum [L.] var White Burley) cells. The genes were transiently expressed 2 to 8 h after transfer of stationary phase cells to fresh medium, during the transition from the quiescent phase of cells leaving the mitotic cycle to the synthesis phase of the cell cycle. After this transient induction, the cells showed a decreased sensitivity to auxin. Although the expression pattern suggests that induction of these genes might be important for cell division, over-production of antisense mRNA for one of these genes (pCNT103) did not influence cell division in transgenic tobacco cells. Furthermore, stimuli such as salicylic acid were capable of inducing gene expression but were unable to restore cell division. Although these data do not conclusively exclude a role for these genes in cell division, their significance in this process is discussed in view of their homology with other auxin-induced genes and in view of the specificity of hormone-induced early responses.

Proteins encoded by an auxin-regulated gene family of tobacco share limited but significant homology with glutathione S-transferases and one member indeed shows in vitro GST activity.

A number of cDNAs corresponding to auxin-regulated mRNAs have been isolated from tobacco and found to be encoded by a multigene family consisting of three subfamilies. Homologous proteins have been isolated independently from soybean and potato. Here we report that the encoded proteins show a limited but significant homology to both plant and animal glutathione S-transferases (GST, EC 2.5.1.18). For the protein NT103, encoded by a member of the Nt103 subfamily, we demonstrate an in vitro GST activity. This is the first time a function is attributed to a member of this group of auxin-induced proteins or any of its homologues. The implications of this finding and the possible relationships between auxins and GSTs are discussed.

Whole-Cell K+ Currents across the Plasma Membrane of Tobacco Protoplasts from Cell-Suspension Cultures.

The whole-cell configuration of the patch clamp technique was used to study both outward and inward ion currents across the plasma membrane of tobacco (Nicotiana tabacum) protoplasts from cell-suspension cultures. The ion currents across the plasma membrane were analyzed by the application of stepwise potential changes from a holding potential or voltage ramps. In all protoplasts, a voltage- and time-dependent outward rectifying current was present. The conductance increased upon depolarization of the membrane potential (to >0 mV) with a sigmoidal time course. The reversal potential of the outward current shifted in the direction of the K+ equilibrium potential upon changing the external K+ concentration. The outward current did not show inactivation. In addition to the outward rectifying current, in about 30% of the protoplasts, a time- and voltage-dependent inward rectifying current was present as well. The inward rectifying current activated upon hyperpolarization of the membrane potential (<-100 mV) with an exponential time course. The reversal potential of the inward conductance under different ionic conditions was close to the K+ equilibrium potential.

Specific transcription and reinitiation of 2,4-D-induced genes in tobacco nuclei.

In order to study the effects of trans-acting factors on cis-acting elements in plant genes an in vitro reinitiating nuclear transcription system is needed. Here we report that run-on experiments with nuclei isolated from 2,4-D-treated auxin-starved early-stationary-phase cells of tobacco clearly show reitintiation of transcription of specific 2,4-D-induced genes. Using gamma-thio nucleotides and [alpha-32P]-UTP we were able to demonstrate the presence of reinitiated labelled specific RNAs after isolation on a mercury-agarose affinity column. Addition of heparin as an inhibitor blocked this reinitiation. In a primer extension assay we found that the new transcripts initiate at approximately the same site as used in vivo.

A non-invasive method for the routine-estimation of fresh weight of cells grown in batch suspension cultures.

A simple apparatus was designed to allow sedimentation of plant cells grown in batch suspensions in Erlenmeyer flasks. After sedimentation the height of the cell mass along the glass wall was measured with a ruler fixed in the apparatus. The cell volume after sedimentation, calculated from this height, appeared highly correlated with the fresh weight of cells. This result was found with eight cell lines in two Laboratories. The method proved to be very suitable to allow routinely measurement of FW without the destruction of cells, from many samples, in a short time, during each phase of the growth cycle.

Promoters of auxin-induced genes from tobacco can lead to auxin-inducible and root tip-specific expression.

In previous studies we have identified several mRNAs which accumulate after addition of 2,4-dichlorophenoxyacetic-acid (2,4-D) to auxin-starved tobacco cells. The mRNAs corresponding to cDNA clone pCNT103 were found to accumulate transiently prior to the cell division response due to auxin treatment. In this study we determined the sequences of three 103-like cDNAs and two 103-like genes, GNT1 and GNT35. To further study the regulation of the expression of these genes their 5' regions were translationally fused with the beta-D-glucuronidase reporter gene (GUS). The GNT1 5' region led to GUS expression only in the root tips of transgenic plants. By using transgenic hairy-root cultures and transformed cell suspension cultures it was shown that the 5' regions of both GNT1 and GNT35 lead to 2,4-D-inducible expression of GUS activity. The homology of the 103-like genes with other auxin-regulated genes is evaluated.

Uptake and accumulation of ajmalicine into isolated vacuoles of cultured cells of Catharanthus roseus (L.) G. Don. and its conversion into serpentine.

Isolated vacuoles from ajmalicine-producing cell suspensions of Catharanthus roseus accumulated the alkaloid ajmalicine. Dissipation of the transtonoplast pH gradient with nigericin abolished ajmalicine accumulation, whereas dissipation of the transtonoplast potential with valinomycin had no effect. Addition of Mg-ATP resulted in a higher ajmalicine accumulation. Serpentine produced by the cells was largely recovered in isolated vacuoles; in contrast, ajmalicine was lost. Ajmalicine was converted in vitro into serpentine by horseradish basic peroxidases (EC 1.11.1.7). In cultured cells there was a striking conformity between the time course of serpentine content and that of the activity of basic peroxidases. Ajmalicine was converted efficiently into serpentine by basic peroxidases extracted from vacuoles and by intact isolated vacuoles. The results are consistent with the hypothesis that ajmalicine accumulates by an ion-trap mechanism and that the accumulated ajmalicine is converted into serpentine inside the vacuoles. By the transformation of ajmalicine into the charged serpentine a trap is created to retain the alkaloids more efficiently in the vacuole.

Effect of auxin on cytodifferentiation and production of quinoline alkaloids in compact globular structures of Cinchona ledgeriana.

Fine cell suspension cultures of Cinchona ledgeriana produce only very low amounts of quinoline alkaloids. These cultures formed self-propagating compact globular structures (CGS) on medium containing 2,4-D and BAP. These CGS could be induced to produce significant amounts of quinoline alkaloids by replacing 2,4-D by low amounts of 1-NAA, which was accompanied by histological changes of the CGS. A few high producing CGS clones could be selected. The stability of this trait was studied over a period of about one year of culture in maintenance medium.

Correlation between Auxin Resistance and the Lack of a Membrane-Bound Auxin Binding Protein and a Root-Specific Peroxidase in Nicotiana tabacum.

The levels of a membrane-bound auxin binding protein (MABP) and a root-specific peroxidase (RSP) were studied in several tobacco (Nicotiana tabacum L.) cell lines including an auxin-resistant variant. Groups of cell lines were distinguished which behaved differentially with respect to MABP and RSP depending on the hormonal composition of the medium. In cell lines in which there existed a correlation between the presence or absence of MABP and that of RSP both phenotypes were expressed if kinetin (1-2 micromolar) was supplied. In contrast, neither MABP nor RSP could be detected under any hormonal conditions tested in the auxin-resistant variant which retains the ability to differentiate shoots but lacks the ability to differentiate roots. About an eightfold increase in the concentration of MABP and a dramatic increase in the activity of RSP occurred in a transformant by a mutant strain of Agrobacterium tumefaciens lacking an intact cytokinin gene when it was grown on medium containing 1 to 2 micromolar kinetin. A correlation between auxin resistance and the lack of MABP and RSP suggests that MABP might be involved in auxin-mediated root differentiation in tobacco.

Auxin-induced rapid changes in translatable mRNAs in tobacco cell suspension.

When 2,4-dichlorophenoxyacetic acid (2,4-D)-dependent tobacco cell suspensions, one normal and one transformed by Agrobacterium tumefaciens, were subcultured on hormone-lacking medium the stationary phase of the cell cycle was reached earlier than on medium containing 2,4-D. Addition of the auxin 2,4-D could restore cell division activity within 10-12 h for the most rapidly reacting cell line. The cell-division response was characterized as being auxin-specific and optimal with 2,4-D at 2.2 10(-6) M. Although the cell lines used showed different characteristics, both reacted with a rapid increase in at least three mRNA species within 1 or 2 h after 2,4-D application. Two, 2,4-D-induced protein spots, seen after in-vitro translation, had the same characteristics (MWs 35 kilodaltons (kDa) and 25 kDa with isoelectric points of 7.1 and 6.3, respectively) in both cell lines. Water-treated controls did not show alterations in the translatable mRNA populations. This indicates that the accumulation of the corresponding mRNAs is an early hormone-induced event. Since cell division is the only measurable reaction found after auxin application, cell systems as described here offer excellent possibilities for studying early auxin-induced changes at the molecular level preceding mitosis.

Auxin-induced mRNA species in tobacco cell cultures.

Using a 2,4-dichlorophenoxyacetic acid (2,4-D)-dependent tobacco cell-suspension culture we analyzed early hormone-induced molecular events preceding cell division. By differential screening of a cDNA library to mRNAs derived from hormone-starved cells treated with 2,4-D for 4 h, seven non-cross hybridizing cDNA clones to 2,4-D-induced mRNAs were obtained. Accumulation of these mRNAs started as early as 15-30 min or less after 2,4-D application. The lowest 2,4-D concentration necessary to induce the mRNAs varied between less than 2.2 × 10(-8) M and 2.2 × 10(-6) M, one mRNA being induced to nearly maximal values at 2.2 × 10(-6) M. Generally, 2,4-D was the most active compound to induce mRNA accumulation, followed by naphthalene-1-acetic acid (NAA). The level of 4 mRNAs increased independently from protein synthesis. Run-off transcription studies showed that the accumulation of some mRNAs was at least partly due to enhanced transcription rates. In different organs of the tobacco plant, the levels of the mRNAs were about as low as in hormone-starved cells. A similar low level of the 2,4-D-induced mRNAs was observed in cells growing in mid-log phase on 2,4-D-containing medium. Only quiescent cells that were triggered to undergo cell division, accumulate these mRNAs transiently.

Naphthylphthalamic acid-binding sites in cultured cells from Nicotiana tabacum.

Naphthylphthalamic acid (NPA), an inhibitor of polar auxin transport, binds with high affinity to membrane preparations from callus and cell suspension cultures derived from Nicotiana tabacum (K d approx. 2·10(-9) M). The concentration of membrane-bound binding sites is higher in cell suspension than in callus cultures. The binding of NPA to these sites seems to be a simple process, in contrast to the binding of the synthetic auxin naphthylacetic acid (1-NAA) to membrane preparations from callus cultures, which is more complex (A.C. Maan et al., 1983, Planta 158, 10-15). Naphthylacetic acid, a number of structurally related compounds and the auxin-transport inhibitor triiodobenzoic acid were all able to compete with NPA for the same binding site with K d values ranging from 10(-6) to 10(-4) M. On the other hand, NPA was not able to displace detectable amounts of NAA from the NAA-binding site. A possible explantation is the existence of two different membrane-bound binding sites, one exclusively for auxins and one for NPA as well as auxins, that differ in concentration. The NPA-binding site is probably an auxin carrier.

Correlation between the presence of membrane-bound auxin binding and root regeneration in cultured tobacco cells.

When cell-suspension cultures and callus tissue from Nicotiana tabacum are grown on medium containing α-naphthaleneacetic acid (NAA) and kinetin, three classes of auxin-binding proteins can be detected. When the herbicide 2,4-dichlorophenoxyacetic acid is used instead of both NAA and kinetin, one of these sites, which is membranebound, disappears. After retransferring cells to medium containing NAA and kinetin, this membrane-bound site reappears after four to eight weeks. This reappearance is correlated with the ability of the cells to regenerate roots.

Cell Division in Nautilocalyx Explants III. Effects of 2,6-dichlorobenzonitrile on Phragmosome, Band of Microtubules and Cytokinesis.

In the highly vacuolated epidermis cells of leaf explants of Nautilocalyx we investigated whether inhibition of cytokinesis by 2,6-dichlorobenzonitrile (DCB) could be caused by a suppression of the formation of phragmosome and band of microtubules (BMT), two structures which are probably involved in cytokinesis and in the determination of the plane of cell division. Interference contrast microscopy showed that DCB (116 µM) did not interfere with the formation of phragmosome and BMT in the expected plane of cell division. Also the positioning of the nucleus and the nuclear division proceeded normally. The phragmoplast was formed, and cell plate formation started, but in most cases the cell plate was not completed. After some time the part of the cell plate already formed shrank: folds appeared, and sometimes tears. The phragmoplast remained present for a long time after this premature end of cell plate growth. Electron microscopical studies showed a shortage of small Golgi vesicles with electron-dense contents in the plane of cell division where very large vesicles with little electron-dense material were present. Furthermore a dilatation of the endoplasmic reticulum in the microtubule zone of the phragmoplast was observed. These results indicate that the DCB-inhibition of cytokinesis does not result from interference with phragmosome and BMT. It seems likely that cytokinesis stops because a weak cell plate is formed that does not mature to a firm cell wall.

A soluble auxin-binding protein from cultured tobacco tissues stimulates RNA synthesis in vitro.

When the soluble auxin receptor from tobacco callus was isolated according to H. Oostrom et al. (1975, FEBS Lett. 59, 194-197; 1980, Planta 149, 44-47) a high polyphenol contamination in the receptor preparation was observed. We developed a new isolation procedure, which drastically reduced this contamination. The receptor, which was partially purified on Sephadex G-200, exhibited the same time- and temperature-dependent binding kinetics as described before (Oostrom et al. 1975, 1980). The Ka for indole-3-acetic acid (IAA) at 25°C was about 1.6·10(8) M(-1) and the number of binding sites varied from 0 to 2·10(-13) M mg(-1) protein. Addition of partially purified receptor preparations to isolated tobaccocallus nuclei resulted in an IAA-dependent stimulation of transcription, which was not observed with similar preparations that did not contain detectable amounts of specific IAA-binding sites. The average stimulation in the presence of 1 µM IAA was 42%; it was achieved by an increase in RNA-polymerase-II activity. The stimulation was not dependent upon the presence of 1 µM IAA during the isolation of the nuclei.

The complex kinetics of auxin-binding to a particulate fraction from tobacco-pith callus.

The kinetics of binding of 1-naphthylacetic acid to particulate fractions from tobacco-pith callus were studied. This binding site does not bind auxin at 0° C. Binding experiments performed at 25° C demonstrated an apparent K a of approx. 6.5·10(6) M(-1). A filtration method was developed in order to study non-equilibrium kinetics of this binding. Dissociation of the complex of auxin and binding site indicates the presence of at least two binding components with dissociation rate constants (k off) of 6.1·10(-3) min(-1) and 6.0·10(-2) min(-1). This binding behaviour was not independent, indicating that the binding of auxin to the particulate fractions was more complex than binding of one hormone molecule to one binding site. This complexity was further confirmed by experiments in which the initial velocity of complex formation was measured. A model was worked out into which our data fit without contradictions. It involves the binding of four hormone molecules to one receptor molecule.

Modulation of the number of membrane-bound auxin-binding sites during the growth of batch-cultured tobacco cells.

We studied the modulation of the number of membrane-bound naphthaleneacetic acid (NAA)-binding sites during the growth cycle of tobacco cells in batch culture. Both cell number and specific NAA-binding increased exponentially, but at different rates and for different periods. This caused a characteristic modulation of the number of binding sites per cell during the growth cycle: During the first day of the lag phase this number decreased; in the exponential phase it rose markedly, and in the stationary phase it was constant.

Characterization of a cytoplasmic auxin receptor from tobacco-pith callus.

Cultured tobacco-pith tissue contains a cytoplasmic receptor for indoleacetic acid (IAA). The concentration of binding sites is very low in comparison to that of several auxin receptors found by other investigators. A few obvious possible causes (degradation or inactivation) were investigated. From the results we conclude that the low number of binding sites is real. The receptor binds IAA optimally at pH 7.5-7.8 and at a temperature of 24-30°C, when incubated for 25-30 min. The binding is very specific, as is shown by competition experiments. The concentration of the receptor in the callus tissue changes dramatically during each culture period, which suggests a possible role in development. The receptor was partly purified by gel filtration on Sepharose 6B followed by ion-exchange chromatography on DEAE-cellulose.