Michael Böttger

Auxin-induced growth and its linkage to potassium channels

We investigated the potassium dependence of rapid auxin-induced growth of scrubbed maize (Zea mays L. cv. Garant) coleoptile segments by means of positional angular transducers. Our setup permitted six simultaneous high-resolution measurements. Provided a buffer with a high concentration of calcium was used in conjunction with well-abraded coleoptiles, (i) there was no auxin-induced growth in the absence of K+ions, whereas in the presence of 1–10 mM KCl auxin-induced growth could be detected after a lag phase of 20 min; (ii) auxin-pretreated plants, which were not growing in the absence of K+ions displayed an instantaneous growth burst if 1–10 mM KCl was added; (iii) this burst of growth did not occur in the absence of auxin, excluding the possibility that it was due to “stored” endogenous growth and (iv) it could not be triggered by Cl-, confirming that it was not an anion effect. The effect was. specific for monovalent cations and showed an apparent selectivity for K+ and Rb+, but was not observed with Li+ or Na+. The action of 1 mM K+ was reversibly blocked by 10–30 mM tetraethylammonium (TEA) and 5 mM Ba2+. Measurements of auxin-induced proton secretion using a computer-controlled pH-stat revealed a similar dependency on the ionic composition of the bathing medium and a similar inhibition by TEA. We suggest that both auxin-induced growth and proton secretion strictly depend on extracellular K+ions and the uptake of K+ mediated by K+ channels at the plasma membrane. These results generally support the acid-growth theory of auxin action, and also concur with evidence that auxin modulation of K+ channels plays a role in the signalling cascade leading to auxin-induced cell elongation.

Oxidoreductases in plant plasma membranes.

Electron transporting oxidoreductases at biological membranes mediate several physiological processes. While such activities are well known and widely accepted as physiologically significant for other biological membranes, oxidoreductase activities found at the plasma membrane of plants are still being neglected. The ubiquity of the oxidoreductases in the plasma membrane suggests that the activity observed is of major importance in fact up to now no plant without redox activity at the plasmalemma is known. Involvement in proton pumping, membrane energization, ion channel regulation, iron reduction, nutrient uptake, signal transduction, and growth regulation has been proposed. However, positive proof for one of the numerous theories about the physiological function of the system is still missing. Evidence for an involvement in signalling and regulation of growth and transport activities at the plasma membrane is strong, but the high activity of the system displayed in some experiments also suggests function in defense against pathogens.

Growth of Avena coleoptiles and pH drop of protoplast suspensions induced by chlorinated indoleacetic acids

Several indoleacetic acids, substituted in the benzene ring, were compared in the Avena straight growth bioassay. 4-Chloroindoleacetic acid, a naturally occurring plant hormone, is one of the strongest hormones in this bioassay. With an optimum at 10-6 mol l-1, it is more active than indoleacetic acid, 2,4-dichlorphenoxyacetic acid and naphthaleneacetic acid. 5-Chloro- and 6-chloroindoleacetic acids are very strong auxins as well. Other derivatives tested have a lower activity. 5,7-Dichloro- and 5-hydroxyindoleacetic acids have very low auxin activity at 10-4 mol l-1 and may be anti-auxins. Some of the derivatives were compared for their effect on pH decline in stem protoplast suspensions of Helianthus annuus L. and Pisum sativum L. The change of pH occurs without a lag period or with only a very short one. Derivatives which are very active in the Avena straight growth assay cause a larger pH decline than indoleacetic acid, while inactive derivatives cause effectively no pH decline.

Apical dominance in roots of Pisum sativum L.

SummaryThe influence of several natural and synthetic hormones on apical dominance of pea roots was investigated. Extracts of roots decapitated just before extraction or 12 h earlier were compared in a lateral root formation assay, in the Amaranthus assay for cytokinins and the Avena coleoptile assay. Indole-3-acetic acid shows a strong promotive effect on the initiation of lateral roots. Gibberellic acid has no significant effect. Abscisic acid and its presumable precursor xanthoxin show a strong inhibitory effect, and so do all cytokinins investigated (kinetin, benzyladenine, and zeatin). Decapitation of roots caused a significant increase in the number of lateral root primordia within 12 h. Paper chromatograms of root extracts indicate the presence of at least two inhibitors of lateral root formation in the alkaline fraction, and of two promoters in the acid fraction. The amount of both inhibitors seems to decrease if the tip of the main root is removed 12 h before extraction whereas the promoters of lateral root formation do not change in their concentration. The Amaranthus test shows cytokinin activity in the region of one of the two inhibitors, and this activity decreases after decapitation. The second inhibitor has the Rf of xanthoxin; it inhibits the growth of Avena coleoptiles and its activity also decreases after decapitation.

Quinones in plant plasma membranes — a missing link?

SummaryThe occurrence of a vitamin-K-like substance (naphthoquinone group) and flavins (flavin mononucleotide and flavin adenine dinucleotide) is demonstrated in plasma membranes isolated from maize (Zea mays L.) roots, on the basis of high-pressure liquid chromotography and spectral analysis. At least three NAD(P)H dehydrogenases could be purified to homogeneity from this plant material. Two of these proteins (25 and 30 kDa) reduce hexacyanoferrate III and quinones, while the third (41 kDa) reduces oxalacetic acid but not hexacyanoferrate III in the presence of NADH. Low-temperature spectra demonstrate the occurrence of a b-type cytochrome in plasma membranes isolated from maize roots. The latter compound could be reduced by ascorbic acid (E0′ > +80 mV) and shows an α-band maximum at 559 nm (at −196 °C). NADH-dependent cytochromeb reduction could be observed only in the presence of detergent and increased after preincubation with vitamin K3 (menadione). On the basis of the presented data a possible function of naphthoquinones in plasma membrane electron transfer is discussed.

Auxin-induced changes in cell wall extensibility of maize roots

Abstract. The rheological properties of corn (Zea mays L. cv. Garant) root elongation zones were investigated by means of a computer-controlled extensiometer. Creep closely followed a logarithmic time function, which was used to quantify creep activity. Pretreatment with auxin, which inhibits extension growth in roots, lowered the creep activity and the apparent plastic extensibility. While the time course of the inhibition of apparent plastic extensibility lagged behind the cessation of elongation growth, the drop in creep activity matched the growth inhibition more closely. Creep activity and apparent plastic extensibility were not significantly affected by pH. These data support the view that the auxin-induced cell wall stiffening (e.g. by cross-linking processes), while causal for the growth inhibition, is not brought about by a cell wall alkalinization.

Proton translocation in corn coleoptiles: ATPase or redox chain?

The O2 dependence of net H+ efflux of maize coleoptiles has been investigated. Below 100 μM O2, H+ efflux in young (1 cm long) coleoptiles is markedly decreased while old (7 cm long) coleoptiles show a decline only at 10 μM O2. Old coleoptiles show the same decrease in net H+ efflux as young ones if treated with fusicoccin. The ratio of alteration of CO2 production to the change in net proton efflux is about 1:1 at 40–80 μM O2 but not at 10 μM O2. An influx can be observed at 10 μM O2 in young as well as in old coleoptiles if the H+ concentration is held at values below pH 6.5. Lower O2 concentrations lead to an increase of net H+ efflux, which might be caused by leaching of organic acids resulting from anaerobic processes, but CO2 production is not significantly changed at these values. It is proposed that more than one system is responsible for proton translocation across the plasmalemma. One of the systems has a high sensitivity to reduced O2 concentration which is within the same range as the high Km of the alternative path.

Levels of Endogenous Indole-3-Acetic Acid and Abscisic Acid During the Course of the Formation of Lateral Roots

Summary The number of lateral primordia arising from roots of Pisum sativum increased significantly 8 hours after decapitation. Abscisic acid inhibited and indoleacetic acid promoted the formation of lateral roots. The level of endogenous abscisic acid decreased to 50 % of its original value; however a change in the level of indoleacetic acid could not be detected. Root tips also contained approximately three times as much abscisic acid as did the basipetal segments. These experiments therefore suggest that abscisic acid is one factor regulating the apical dominance of roots.

The effect of ascorbate free radical on the energy state of the plasma membrane of onion (Allium cepa L.) root cells: alteration of K+ efflux by ascorbate?

In this work we demonstrate the effect of different ascorbate redox states on the plasma membrane potential and on proton secretion of onion root cells. Ascorbate and dehydroascorbate induced plasmalemma hyperpolarization and stimulated acidification of the incubation medium. Both effects were transient and a repolarization of the membrane to the resting potential was reached within 10–15 min. The free radical of ascorbate had a permanent stimulatory effect on both parameters: a concentration dependent hyperpolarization of about 25 mV, partially inhibited by vanadate, could be observed immediately after addition. Medium acidification was increased by a factor of 20 and sustained for hours in the presence of the free radical. Additionally, ascorbate may increase the potassium permeability of the membrane, thus hyperpolarizing at low external potassium concentrations. Possible interactions of the free radical with the energization of the plasma membrane via an NAD(P)H-ascorbate free radical reductase (EC 1.6.5.4) will be discussed.

The temporal correlation of changes in apoplast pH and growth rate in maize coleoptile segments

Auxin induces extracellular acidification in growing shoot tissue. The causal relationship between this process and auxin-mediated growth is debated, partly because of contradicting previous reports on the temporal correlation of auxin-induced apoplast pH-drops and growth bursts. We have simultaneously measured both parameters on the background of spontaneously occurring endogenous changes in growth rate and apoplast pH in maize coleoptile segments. Our data demonstrate good temporal correlation, during both the ‘Spontaneous Growth Response’ and the response to exogenous auxin, which is transient under the conditions chosen due to rapid auxin metabolism. We suggest that cell wall pH and growth rate are co-regulated in this organ, and that contradictions in the literature might be due to technical difficulties.