Johannes Jung

Plant Growth Retardants as Inhibitors of Ethylene Production

Summary Plant growth retardants of the norbornanodiazetine (tetcyclacis) and triazole (BAS 111 W) types inhibited ethylene production in leaf discs of barley and oilseed rape. The maximal reduction of 70 % was reached 5 h after treatment. The addition of gibberellic acid could not overcome the effects. At concentrations of the retardants up to 3 x 10-4 mol x 1-1 the reduction in ethylene formation was accompanied by increased or nearly constant levels of 1-aminocyclopropane-l-carboxylic acid (ACC) and N-malonylACC in the leaf discs. This suggests an inhibition of the conversion of ACC to ethylene in the biosynthetic pathway. As explanations a direct influence on the ethylene-forming enzyme or an indirect effect via a retardant-caused modification in the cellular membrane properties are discussed.

Influence of the growth retardant tetcyclacis on cell division and cell elongation in plants and cell cultures of sunflower, soybean, and maize.

Taking tetcyclacis, a norbornenodiazentine derivative, as an example, the influence of a growth retardant on the shoot growth of sunflower, soybean, and maize seedlings grown and treated in hydroculture was investigated. In detail, the reduction in the length of various shoot sections {epicotyl, 1st internode, leaf blade) caused by the retardant was studied. At low concentrations of the retardant (\lt10(-6) M) the shortening effects are substantially attributable to an influence on cell elongation, whereas cell division is inhibited as the concentration increases (τ10(-6) M). A comparison of the effects of tetcyclacis in cell suspension cultures of appropriate plant species showed that also in this system concentrations τ 10(-6) M inhibited cell division growth, i. e. there is comparability of plant/ cell culture regarding the retardant effect on cell division. In contrast to the intact plants, however, cell elongation appears to be of only subordinate importance for the growth of cell cultures, as it has been shown using parsley cell suspension cultures. It is discussed to what extent influencing the gibberellin or sterol biosynthesis by means of tetcyclacis provides an explanation for the concentration-dependent effect on the cell division and cell elongation processes.

Changes in membrane permeability and mineral, phytohormone and polypeptide composition in rice suspension cells during growth and under the influence of the growth retardant tetcyclacis

The plant growth retardant tetcyclacis inhibits cell division growth in rice suspension cultures at concentrations above 10−6 M. Tracer experiments with rice cells revealed that tetcyclacis reduced the incorporation of mevalonic acid into terpenoids after 30 min, the uptake of leucine, uridine and thymidine after 2 h and their incorporation into the corresponding macromolecules after 3–7 h. The changes in membrane permeability concluded to have been caused by an influence on phytosterol biosynthesis are probably also the explanation for alterations of tetcyclacis-treated cells in the content of macro- and microelements.As shown by immunoassay, tetcyclacis did not modify the levels of endogenous gibberellins (Grossmann et al. 1985), cytokinins and indole acetic acid during a growth cycle of 15 d. However, a clear rise in the abscisic acid (ABA) level occurred during the first 5 d of treatment. In untreated cells such a rise coincided only with the aging of the cell culture in the stationary growth phase. Investigations of the cell polypeptide pattern using sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed that the ABA increase following tetcyclacis treatment seems not to be a consequence of advanced cell aging.

Effects of NDA, a new plant growth retardant, on cell culture growth ofZea mays L.

A new plant growth retardant, the norbornenodiazetine derivative 5-(4-chlorophenyl) - 3,4,5,9,10 - pentaaza - tetracyclo - 5,4,1,02.6,08.11- dodeca - 3,9 - diene (NDA) was tested for its effects on growth ofZea mays suspension cultures. It was shown that NDA could inhibit cell division almost completely at a concentration of 5× 10−5 M, while 80% of cells could be considered viable. Tracer experiments revealed that NDA inhibited thymidine, uridine, and leucine uptake into cells after 30 min of application. In contrast, amino acid incorporation into proteins was reduced only after one day of treatment and incorporation of precursors into DNA and RNA still later. Since NDA stimulated DNase, RNase, and protease activity in the cells simultaneously, an enhancement of DNA and RNA in cells possibly was prevented. That NDA affected protein synthesis indirectly seemed to be proved by the late point in time of its action on leucine incorporation and by only slight effects on cell free translation. An explanation of these findings could be an alteration in or inhibition of sterol biosynthesis caused by NDA, because it is known that sterols play an important role in controlling permeability of plant membranes as well as in maintaining normal protein synthesis. Thus we tested NDA for its effects on sterol production in maize cells and demonstrated that the composition of the sterol fraction, mainly stigmasterol and β-sitosterol, was clearly changed qualitatively as well as quantitatively.

Effectiveness of new terpenoid derivatives, abscisic acid and its methyl ester on transpiration and leaf senescence of barley

Summary The effectiveness of the new terpenoid derivatives LAB 144 143 and LAB 173 711 in reducing water loss through transpiration in barley ( Hordeum vulgare L Cv. Union) was compared with that of abscisic acid (ABA) and ABA methyl ester. The compounds were applied in biotest systems using detached primary leaves, as well as sprayed on intact greenhouse-grown barley plants. If the compounds were applied to isolated leaves via the transpiration stream all substances reduced water consumption to nearly the same substantial extent. With the foliar treatment of detached leaves LAB 144 143, LAB 173 711 and ABA methyl ester, in the order of decreasing activity, were slightly more active than ABA. This result was paralleled by the capacity of the compounds to close the stomata. In intact barley plants, too, LAB 144 143 was the most effective antitranspirant tested. It is speculated that especially LAB 144 143 penetrates the leaf surface of barley better than the other compounds. Furthermore, the new terpenoid derivatives promoted the senescence process of barley leaf segments in the same way as ABA did. Considering the structural relationships of the new compounds to ABA and their biological spectrum of action it is suggested that LAB 144 143 and LAB 173 711 are functional analogues of ABA.

Transpiration-inhibiting abscisic acid analogs

Synthetic analogs of abscisic acid (ABA) and their inhibiting effect on transpiration rates of detached barley leaves are presented. By systematically varying the carbon skeleton of ABA, the influence of structural changes on biological activity was investigated. The results show that a properly substituted cyclohexane unit and a six-carbon side chain seem to be indispensable for high ABA-like activity, whereas the oxidation state of the terminal carbon atom in the side chain appears to be less essential. Thus, synthetic compounds have been created that exhibit biological activities comparable both in type and strength with ABA itself. On the basis of molecular models, a hypothesis of the geometric arrangement of essential substructural units is proposed.

Is ethylene involved in the regulation of growth of sunflower cell suspension cultures

Summary Undifferentiated, 2,4-dichlorophenoxyacetic acid (2,4-D)-dependent suspension cells of sunflower ( Heliantbus annuus L. Spanners Allzweck) produce large quantities of ethylene during the exponential growth phase with a maximal rate of 2 nmoles ethylene per 10 6 cells h −1 . A comparison of the effects evoked by a broad range of compounds interfering with growth and/ or ethylene biosynthesis revealed a positive correlation between the rate of cell division and ethylene formation. Aminoethoxyvinylglycine (AVG) inhibited growth parallel to the ethylene production. The addition of aminocyclopropane carboxylic acid (ACC) partially reversed the decrease in ethylene synthesis and the effect on growth. Data suggest that this partial compensation of the inhibition in cell division is attributable to a reduction in AVG-uptake caused by simultaneously applied ACC. Addition of 2,4-D to 2,4-D-deprived cultures caused a maximum rise in growth at 10 −6 mol × 1 −1 and in ethylene synthesis at 10 −5 mol × 1 −1 . Thus, 2,4-D in a concentration of 10 −7 mol × 1 −1 strongly stimulated growth without an increase in ethylene formation. This indicates that 2,4-D stimulates ethylene production independently of its effect on growth. The results provide strong evidence that in sunflower suspension cultures there is no functional relationship between growth and ethylene synthesis. It is assumed that ethylene is a by-product of actively dividing suspension cells.

Plant cell suspension cultures as model systems for investigating growth regulating compounds

Several plant growth regulators were investigated for their activity in cell suspension cultures of Glycine max, Gossypium hirsutum and Zea mays. The effect on the growth of the cell cultures was traced by means of cell counting and determining packed cell volume and turbidity of the suspensions. The growth retardant 5-(4-chlorophenyl)-3,4,5,9,10-pentaaza-tetracyclo-5,4,102,6 ,08,11-dodeca-3,9-diene (NDA) and, to a slightly lesser extent, ancymidol proved to be the compounds with the greatest inhibitory action on cell division growth of all three cell cultures. In the case of cotton this effect was accompanied by increased synthesis and secretion of cell-wall material. Staining methods showed that, especially in the case of NDA, a high percentage of cells could be considered as viable, and showed thus that NDA inhibits the cell division process while the cells remain metabolically active. The effects of 1,1-Dimethyl-piperidiniumchloride (DPC), a genuine growth retardant of cell propagation, and, with less efficiency, N-trimethyl-(β-chloroethyl)-ammoniumchloride (CCC) in cotton, the triazole LAB 117 682 in soybean and maize, and, to a lesser extent, (2-isopropyl-5-methyl-4-trimethyl-ammoniumchloride)-phenyl-l-piperidiniumcarboxylate (AM0-1618) in soybean can be regarded as species-specific. Otherwise, CCC and particularly daminozide exhibited no action at the concentrations used. A comparison of the data from hydroculture studies with soybean and maize seedlings showed considerable agreement with the effectiveness of the substances in the corresponding cell cultures. Thus, cell cultures can be used to identify and screen substances with growth-influencing activity, and may also offer new ways to elucidate the mode of action of plant growth regulators.

Inhibition of ethylene production in sunflower cell suspensions by a novel oxime ether derivative.

During the incubation of undifferentiated cell suspensions of sunflower (Helianthus annuus L. cv. Spanners Allzweck) ethylene production was effectively inhibited by the novel oxime ether derivative LAB 181 508, [[(Isopropyliden)-amino]-oxy]-acetic acid-2-(methoxyl)-2-oxoethylester (PACME). The compound was most active during the first 6 days of incubation exhibiting a value of 50% inhibition at 9.5×10−6 mol×L−1. The pattern of changes in the internal 1-aminocyclopropanecarboxylic acid (ACC) and N-malonyl-ACC (MACC) levels paralleled the influence on ethylene formation. While the addition of ACC fully restored ethylene production, applied S-adenosyl-L-methionine (SAM) was not effective. Experiments with [14C]indole-3-acetic acid (IAA) revealed that LAB 181 508 did not affect auxin uptake into suspension cells of sunflower. The results suggest that LAB 181 508 reduces ethylene formation by inhibiting the conversion of SAM to ACC in the biosynthetic pathway. In comparison to the structurally related inhibitor of ACC synthase, aminoethoxyvinylglycine (AVG), LAB 181 508 reduced growth and viability of the suspension cells only slightly. Low phytotoxicity of LAB 181 508 combined with a less complicated chemical synthesis might offer interesting aspects for physiological research and horticultural and agricultural practice.

Accumulation of Abscisic Acid in Cell Suspension Cultures of Oilseed Rape Treated with the Growth Retardant BAS 111 .. W: Effects on Osmotic Potential and Potassium, Water and Sugar Contents

Summary Cell suspension cultures of oilseed rape ( Brassica napus L. cv. Kasan) showed a transient increase in cellular abscisic acid (ABA) content at the beginning of the exponential growth phase, with a maximum after 5 days of subculture. In proportion to the endogenous levels, ABA was excreted into the culture medium. Treatment with 10 -4 mol·L -1 fluridone, an inhibitor of ABA biosynthesis, suppressed the rise in ABA levels almost completely. Thus, ABA might be produced in response to osmotic stress resulting from subculturing the cells in fresh medium with higher sugar and mineral concentrations. This hypothesis was supported by the finding that endogenous ABA accumulation correlated with increasing osmotic stress induced by mannitol (from -0.39 to -0.74MPa) in the medium. Concomitantly, cellular water content and the osmotic potential of the cell sap decreased. Application of the triazole-type growth retardant BAS 111 .. W also increased the amount of endogenous ABA, but without changes in the osmotic potential of the medium. The compound is thought to inhibit ABA catabolism to phaseic acid. In contrast, the cyclohexanetrione-type retardants prohexadione calcium and LAB 198 999 did not alter ABA contents significantly. Dose response curves obtained 5 days after treatment with BAS 111 .. W revealed that ABA accumulation in the cells was accompanied by rising potassium and water levels and osmotic potential of the cell sap. The latter effect could be explained by a restricted uptake of sucrose and/or its hydrolysis products glucose and fructose, in BAS 111 .. W treated suspension cells. Calculated on a cell number and cell dry weight basis, ABA, potassium and water content increased to approximately 210, 160 and 140%, respectively, of the control values after treatment with 10 -5 to 10 -4 mol·L -1 BAS 111.. W. A significant increase was also found in magnesium while calcium was only slightly elevated and sodium levels remained unchanged. The changes in the potassium and water contents following the elevated endogenous ABA levels mediated by BAS 111 .. W were thus opposite to those observed when ABA was increased in response to osmotic stress. The specific effects of ABA on these parameters may thus be elucidated without interference from the effects of simultaneous water stress. It is speculated that retardant-caused ABA accumulation influences endogenous potassium, water and sucrose levels and the osmotic potential of the cell sap.