K. R. Libbenga

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 1.6·108 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.

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.

A high affinity receptor for indoleacetic acid in cultured tobacco pith explants

The mechanism of action of auxins in long-term responses of plant tissue to these hormones is still unknown. However, it is very likely that the first event in the chain of processes leading to, for instance, cell division or differentiation, is coupling of the auxin with a cellular binding site. It is therefore logical to start an investigation on the mechanism of action of indoleacetic acid (IAA) with a study of its primary binding site(s) or receptor(s). Such a receptor should meet criteria derived from physiological effects. It should have a high affinity (association constant K, = 1 O7 ~1 OS M-’ ) and a low capacity; the auxins which are bound should probably show structure--binding relations similar to the structure--activity relations in physiological responses. The localization and characteristics of the receptor depend, of course, very much on its function in the cell. The work presented here is based on the assumption that this function is performed in the nucleus. Therefore, we mainly investigated the cytosol fraction and not the membrane fraction of our extracts, for localization of the receptor in the plasma membrane would probably mean that a second messenger is involved in its function. In this paper we present evidence that a receptor for IAA with the expected properties does exist, and is localized in the cytosot fraction of tissue cultures derived from tobacco stem pith. Investigations concerning the function of this receptor in the cell are in progress. 2. Material and methods

A particle-bound auxin receptor from tobacco pith callus

Abstract The binding of NAA to particulate fractions isolated from tobacco pith callus was studied. The presence of a specific auxin receptor with high affinity was demonstrated. NAA-binding was maximal after 30 min of incubation at 36°C (pH 5). The affinity constant for NAA was approx. 3 × 106/mol and the concentration of binding-sites was approx. 75 pmol/g fresh weight (60 pmol/mg protein). The affinities for various auxin analogues were roughly correlated with their activities in auxin bio-assays. The same type of receptor was found in freshly excised tobacco pith, but at a lower concentration.

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.

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.

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 Ka of approx. 6.5·106 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 (koff) 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.

Transcription in nuclei isolated from tobacco tissues.

An advantage of using isolated nuclei for the study of transcription is the presence of endogenous RNA polymerases. Moreover, in the nuclei, the native state of the chromatin, including regulatory proteins, is maintained. Transcription in nuclei from various plant sources, e.g., soybean hypocotyls [ 1 ] and tobacco callus [2] has been studied. Using tobacco callus nuclei as an in vitro system we did not find any effect of hormones on transcription [3]. However, one should be aware that nuclei isolated from different tissues of the same plant species may behave differently. Therefore, we have compared the characteristics of nuclei isolated from tobacco stem pith, pith callus and leaves. Clear differences between the properties of the nuclei were found in the amounts of RNA synthesized per Irg DNA, in the contents of free RNA polymerases, and in the stimulation of RNA synthesis by heparin. Exogenous DNA of various origins was transcribed to a different extent by the nuclei studied.

Auxin binding site in tobacco cells

A specific, high affinity, IAA binding site was demonstrated in both a cytosolic fraction, and in isolated nuclei, fromNicotiana tabacum cv. Wisconsin No. 38 cells grown in suspension culture. The amount of the binding site detected in both these fractions changed during the culture cycle according to a strict pattern. The molecular mass of the binding site was estimated by gel filtration to be approximately 175 000 and it appears to be a protein. When partially purified by affinity chromatography and allowed to pre-incubate with IAA, the site had a significant stimulatory effect on total RNA synthesis, as measured by a cell-free assay system. Unpurified extracts had no such effects. The system behaves rather like the steroid hormone-receptor system in animals.

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 (Kd 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 Kd 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.