Interaction of the plant hormone, indole-3-acetic acid, with phosphatidylcholine model membranes: effects of acyl chain length.

The interaction between the plant hormone, indole-3-acetic acid (IAA), and phosphatidylcholines (PC) of varying acyl chain length has been studied by monitoring the IAA-induced changes in 1H-NMR chemical shifts of lipid headgroup -+N(CH3)3 protons. For PCs in both micellar and vesicle bilayer systems these shifts increase with chain length although for the latter the magnitude of the shifts decreases with an increase in chain unsaturation. In systems composed of mixtures of pure PCs the headgroup -+N(CH3)3 resonance for each phospholipid is shifted by IAA to different extents, indicating that IAA is able to distinguish between individual PCs in mixtures. In di-C12PC and di-C14PC, but not di-C10PC vesicle systems, the -+N(CH3)3 resonance is split into two components reflecting differences in packing of the inside and outside lamellae. This splitting is altered by IAA indicating that IAA interacts differently with the inside and outside PC molecules.

Elimination of self-association as the source of the thermodynamic nonideality in aqueous proline solutions.

The effect of high concentrations of proline on the diffusion coefficient of water has been examined to assess the extent to which the resulting thermodynamic nonideality could be explained on the statistical-mechanical basis of excluded volume. In fact, such a space-filling role not only accounts for the proline concentration-dependence of the diffusion coefficient of water but it also accounts for the nonideality of proline in freezing point depression and isopiestic measurements. These findings refute the conclusion (Schobert, B. and Tschesche, H. (1978) Biochim. Biophys. Acta 541, 270-277) that the stabilization of enzyme structure by high concentrations of proline stems from self-association of the imino acid via intermolecular hydrogen bonding; and thereby support the concept that the protective effect of proline on enzyme stability must reside mainly in its action as an inert, space-filling solute.

Indole-3-acetic acid-mediated transport of Mn2+ and other ions across phosphatidylinositol vesicular membranes as determined by 31P-NMR.

The indolic plant hormone, indole-3-acetic acid (IAA), mediated the transport of Mn2+ and other ions into small unilamellar vesicles prepared from soybean phosphatidylinositol (PI) and this process has been studied using 31P nuclear magnetic resonance spectroscopy (NMR). The rate of Mn2+ movement into PI vesicles is dependent on IAA concentration and temperature with an IAA stoichiometry of 4.1 and an activation energy of 16.8 kcal mol-1 derived for the rate-determining process. These values are altered by low concentrations of endogenous ions (which can be removed by treatment with EDTA) present in the PI. With non-EDTA-treated PI, values of 2.3 and 23.0 kcal mol-1 were obtained for the stoichiometry and activation energy, respectively. These values indicate that (a) IAA interacts with PI membranes; (b) IAA-induced changes in membrane permeability can be substantially modulated by ions and (c) IAA very significantly influences the rate of movement of some (but possibly not all) cations across PI membranes. Such effects are also modified by the oxidation state of the PI.

Plant growth retardants as inhibitors of sterol biosynthesis in tobacco seedlings.

Three plant-growth retardants 2'-isopropy1-4'-(trimethylammonium chloride)-5-methylphenylpiperidine carboxylate (Amo 1618), beta-chloroethyltrimethylammonium chloride, and tributyl-2, 4-dichlorobenzylphosphonium chloride were tested for their effects on sterol production in, and growth of tobacco (Nicotiana tabacum) seedlings. As the concentration of each retardant increased, there was an increased inhibition of the incorporation of dl-2-(14)C-mevalonic acid into sterol (particularly desmethylsterol) fractions and an increased retardation of stem growth. Growth retardation was observed with both single and repeated retardant treatments, and with Amo 1618, in particular, a close quantitative relationship between inhibition of sterol biosynthesis and stem growth was obtained. Gibberellic acid completely overcame retardant effects and application of sterols also restored normal growth. It is concluded that the concept of causality in the relationship between growth retardation and gibberellin biosynthesis is probably premature, since growth retardants have a more general inhibitory action on isoprenoid biosynthesis in plants.

Field control of plant growth and development through the laser activation of phytochrome.

Helium-neon lasers emitting at 632.8 nm can influence the phytochrome controlled growth and development of plants more than a quarter of a mile away. It may therefore be possible to control the growth of crops using lasers to activate the phytochrome system.

Physiological Effects of Gibberellic Acid. IX. Recovery of Gibberellic Acid Following Incubation with Endosperm.

To determine the fate of gibberellic acid (GA(3)), solutions were incubated for 24 hours with or without barley endosperm and were subsequently applied to dwarf maize seedlings. Hormone activity appeared to increase as a result of incubation with endosperm. This apparent increase in GA(3) concentration was probably due to a synergistic interaction between GA(3) and endosperm constituents, particularly carbohydrate, released during the incubation period. It is concluded that relatively little hormone is inactivated during the initiation of endosperm mobilization.

Physiological effects of gibberellic Acid. X. The release of gibberellin-like substances by germinating barley embryos.

Barley embryos, completely free from endosperm, were excised from germinating grain at various times and allowed to diffuse into an aqueous medium for varying lengths of time. At the end of this time, the embryos and ambient solutions were separately extracted. Gibberellin-like activity in the extracts was determined with the barley endosperm bioassay using seed from the same variety, harvest and treatment schedule as was employed for the embryo diffusion experiments. Gibberellin-like substances were released by embryos throughout the 60 hour germination period, though at no time during this period could sufficient activity be extracted from the embryos themselves to account for the observed release. Solvent partitioning and chromatography identified at least one major acidic component migrating at an Rf similar to that of GA(3).It is concluded that the endogenous gibberellin-like substance(s) originates within the embryo during germination, and that the release of this substance(s) is temporally consistent with, and quantitatively sufficient to account for the in vivo endosperm mobilization response syndrome. A gibberellin-like substance is undoubtedly the endosperm mobilizing hormone.

Inhibition of Sterol Biosynthesis by 2-Isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine Carboxylate Methyl Chloride in Tobacco and Rat Liver Preparations.

2-Isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine carboxylate methyl chloride, 90%, applied to rootless tobacco (Nicotiana tabacum cv. Samson) seedlings inhibits the incorporation of (14)C-mevalonate into sterols. Concomitantly, the retardant causes the accumulation of squalene-2,3-epoxide, an intermediate in sterol biosynthesis. The results with tobacco are identical to those produced by the retardant in cell-free rat liver preparations.

The influence of gibberellic Acid on the permeability of model membrane systems.

Gibberellic acid increases the permeability of model membranes composed of various plant-source lipids, a sterol, and dicetyl phosphate. As a result of hormone treatment, the flux of uncharged molecules such as glucose or sucrose, or charged ions such as chromate, through the model membranes (liposomes or micelles) is increased. The revelance of this finding to the in vivo effects of the hormone is briefly discussed.

Comparison of IAA-Induced and Low Temperature-Induced GA(3) Responsiveness and alpha-Amylase Production by GA(3) Insensitive Dwarf Wheat Aleurone.

Rht3-containing gibberellic acid (GA(3)) insensitive deembryonated wheat (Triticum aestivum L. var Cappelle Desprez x Minister Dwarf) aleurone, that can be made responsive to GA(3) by low temperature, can also be rendered GA(3) sensitive by preincubation with indoleacetic acid (IAA). The IAA-induced response of the dwarf selection is concentration-dependent, relatively sensitive, and similar in magnitude to that induced by low temperature. Other auxins also induce GA(3) responsiveness to a greater or lesser degree. IAA has no apparent effect on the wild type (rht, tall) selection.

A Possible Role for Indoleacetic Acid, Low Temperature, and Phospholipid Metabolism in the Induction of GA(3) Responsiveness in GA(3) Insensitive (Rht3-Containing) Dwarf Wheat Aleurone.

Preincubation of dwarf, Rht3-containing deembryonated seed for 4 hours in 342 nanomolar indoleacetic acid (IAA) induced maximum sensitivity to GA(3). In addition, the 4-hour IAA pretreatment caused a 2-fold increase in total phospholipids which coincided identically on a temporal basis with the induced GA(3) sensitivity. Changes in absolute levels of individual phospholipids and their acyl groups were recorded and compared with the changes observed in several Rht-containing aleurone tissues which were induced to develop GA(3) sensitivity by exposure to low temperature (5 degrees C). Several distinct similarities between all tissues were recorded as they develop GA(3) sensitivity. One parameter, the percentage phospholipid composition, was quite similar in all tissues after they had become maximally sensitive to GA(3), suggesting that there is at least one membrane phospholipid composition which is particularly responsive to GA(3). The results indicate that (a) the basis of the GA(3) insensitivity of the Rht mutation resides in an aberrant phospholipid/fatty acid composition and/or metabolism; (b) exposure to low temperature (5 degrees C) for 20 hours or longer, or 342 nanomolar IAA for 4 hours or longer reverses or corrects the genetic lesion, enabling the tissue to adopt a GA(3) responsive membrane composition. Finally, an hypothesis is discussed which indicates that IAA may play a controlling role in the mobilization of endospermal reserves, at least in Rht3-containing wheat aleurone.

Purification of gibberellic acid-induced lysosomes from wheat aleurone cells.

Using isopycnic density gradient centrifugation, lysosomes were concentrated in a single region of a sucrose-Ficoll gradient (p = 1-10 g cm-3), well separated from most other cell organelles. Gibberellic acid-induced lysosomes were found to be rich in alpha-amylase and protease but not ribonuclease. The lysosomal band also contained a majority of the NADH2-cytochrome c reductase, a marker enzyme for endoplasmic reticulum, found in the gradient. Examination of electron micrographs revealed that a purified band of lyosomes contained at least 3 vesicle types, ranging in size from 0-1 to 0-5 mum. The significance of these findings to proposed mechanisms of action of gibberellic acid is discussed.

Lysosomal nature of hormonally induced enzymes in wheat aleurone cells.

The subcellular distribution of the enzymes alpha-amylase, protease and ribonuclease in wheat aleurone layers after treatment with gibberellic acid was determined by differential centrifugation. Of the alpha-amylase 56% was precipitable from cell homogenates, indicating that it is a particulate enzyme. Similar results were recorded with protease. Particulate alpha-amylase showed distinct structural latency, and membrane-rupturing mechanical or chemical treatments were required to release the enzyme in an active form; the results were completely analogous to results with lysosomal enzymes found in animal tissues. The identification of the hormonally induced enzymes as lysosomal suggests that the hormonal mechanism may be more closely associated with extracellular enzyme synthesis rather than with nucleic acid metabolism.

Low temperature induction of hormonal sensitivity in genotypically gibberellic Acid-insensitive aleurone tissue.

An exposure of genetically gibberellic acid-insensitive isolated wheat aleurone tissue/deembryonated seeds to low temperature for 20 hours prior to addition of exogenous gibberellic acid results in a significant increase in sensitivity to gibberellic acid. The results may reflect a low temperature-induced increase in hormone receptor sites and could have important implications for elucidating the nature of the primary site of hormone action.

Low Temperature-Induced GA(3) Sensitivity of Wheat : I. Characterization of the Low Temperature Effect on Isolated Aleurone OF KITE.

Gibberellic acid (GA(3)) sensitivity (measured as alpha-amylase production) of the isolated aleurone tissue/deembryonated seed of two wheat (Triticum aestivum L. var Kite and var Aroona) varieties each containing either one of the dwarfing genes, Rht1 and Rht2, was increased significantly as a result of low temperature treatment. The magnitude of the low temperature-induced increase occurred without any change in the lag time of alpha-amylase production. This low temperature induction of GA(3) sensitivity was found to be operative in aleurone tissue of only those varieties having at least one of the three Rht alleles. It is likely, therefore, that the low temperature treatment effect which ;cures' or circumvents the genetic lesions manifest in the Rht1 and Rht2 genotypes is the same as that effective in the Rht3-containing genotype and probably involves an increase in hormone (GA(3)) receptor sites. Furthermore, this increase appears to be a quantitative temporal one.

Low Temperature-Induced GA(3) Sensitivity of Wheat : II. Changes in Lipids Associated with the Low Temperature-Induced GA(3) Sensisivity of Isolated Aleurone of Kite.

Exposure of isolated aleurone tissue from the wheat (Triticum aestivum) variety Kite which contains the Rht2 allele, to low temperature (5 degrees C) for 20 h prior to addition of exogenous GA(3), resulted in significant changes in the content of lipids, especially phospholipids. Significant low temperature-induced changes in both the head group and acyl contents of two phospholipids, phosphatidylcholine and phosphatidylethanolamine, were detected. More importantly, these changes displayed a very close temporal relationship with the low temperature-induced increase in GA(3) sensitivity. Further, this relationship was paralleled by a highly significant correlation between the changes in the phospholipids and the changes in alpha-amylase production. These results underline the possibility that the GA(3) receptor sites are membrane-based lipids.

Delta-Pyrroline-5-carboxylic Acid Dehydrogenase in Barley, a Proline-accumulating Species.

The characteristics of the enzyme Delta(1)-pyrroline-5-carboxylic acid dehydrogenase from etiolated barley (Hordeum distichum) shoots have been examined. The bulk of the enzyme activity was found in the 10,000g pellet fraction, this activity being displayed only after detergent treatment of the suspended pellet. The enzyme was most active at pH 8, and activity was NAD-dependent. Enzyme activity was unaffected by either mannitol or sucrose in the reaction mixture up to a concentration of 0.45 m but was strongly inhibited by Cl(-) and, to a lesser extent, SO(4) (2-). The inhibition attributable to KCl was reversed by increasing the concentration of Delta(1)-pyrroline-5-carboxylic acid in the reaction mixture.

Barley endosperm bioassay for gibberellins. I. Parameters of the response system.

Parameters of the bioassay based on the gibberellin-induced reducing sugar release of barley endosperm were investigated. Procedures for the rapid handling and processing of up to several hundred treatments without loss in sensitivity of the test are described, and the effects of variations in many aspects of the bioassay were assessed.In general, the variations in varieties, techniques, additives, conditions, and even gibberellins, all illustrate the stability, sensitivity, and adaptability of the hormone-induced response and emphasize its utility as a gibberellin bioassay.

Barley Endosperm Bioassay for Gibberellins. II. Application of the Method.

The use of the modified endosperm bioassay in conjunction with techniques for extracting and paper chromatographing extracts, and the application of statistical analyses to the results, is reported. The modified procedure has not altered the relative response to different gibberellins, though an analysis of many standard curves indicates that at least 1 feature of the bioassay system is still uncontrolled. A comparison of this bioassay with 15 others indicates that the endosperm response may have wide applicability in explorations of the physiological effects of gibberellins.