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.

Rationalization of the effects of compatible solutes on protein stability in terms of thermodynamic nonideality.

Inhibition by compatible solutes such as proline and glycine betaine of the rate of coagulation, at 60 degrees C, of bovine serum albumin in 0.1 M acetate buffer, pH 5, is used as a model system to substantiate the concept that the production of high concentrations of osmolytes by plants and other organisms in response to stress (e.g., drought) results in stabilization of native enzyme structures via nonspecific excluded volume effects. The paradoxical situation whereby this effect of compatible solutes counters to some extent the protein-precipitating effect of poly(ethylene glycol) is also seemingly resolved.

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.

Variability in Proline-Accumulating Ability of Barley (Hordeum vulgare L.) Cultivars Induced by Vapor Pressure Deficit.

This work was undertaken in an effort to reconcile the conflicting proline-accumulating responses of the barley (Hordeum vulgare L.) cultivars, Excelsior and Proctor, reported by Singh et al. (1972) and Hanson et al. (1976). It deals with the effects of different vapor pressure deficits (VPD) during growth and subsequent drought stress on several barley cultivars. A higher VPD (1.2 kilopascals) during Clipper seedling growth resulted in higher solute-accumulating ability, seemingly independently of leaf water potential, than a lower VPD (0.12 kilopascals). The higher VPD during stress also resulted in higher solute contents, and this response may be more closely related to leaf water potential. When the responses of Excelsior and Proctor were examined in detail, it was found that the relative proline-accumulating ability of the two cultivars was dependent upon the VPD under which they were grown. At low VPD, Proctor accumulated significantly more proline than did Excelsior; whereas at higher VPD, Excelsior accumulated more proline than did Proctor. The crossover occurred at a VPD of about 0.72 kilopascals. This reversal of cultivar response was enhanced by multiplying seed under the two VPD extremes. Glycinebetaine accumulation did not demonstrate the crossover effect, although the concentration of this compound in all cultivars also depended on the VPD prevailing during growth and/or stress. Solute levels, in general, were more closely related to the decrease in relative water content than to a decrease in leaf water potential. It is concluded that the conflicting proline-accumulating responses of Excelsior and Proctor could be explained by these findings.

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.

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.

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.

Proline and glycine betaine influence protein solvation.

Glutamine synthetase from barley (Hordeum distichum L.) is precipitated by polyethylene glycol (PEG). Proline, in a concentration-dependent manner, reduces the amount of enzyme precipitated by PEG, although the effect of the imino acid can be counteracted by raising the level of PEG. The effect of PEG is a function of mer number and concentration and the influence of both elements can be ameliorated by proline. PEG-induced enzyme precipitation is a function of pH, as is its interaction with both proline and betaine in the reaction. The lack of effect of amount of enzyme on the proline and PEG effects supports the conclusion that, in this system, proline and PEG do not function through interaction with the protein. Other compounds, such as glycine, glucose, and sucrose, can decrease the PEG-induced precipitation of the enzyme, although glycerol was not active under the conditions employed.The results are consistent with the proposition that a protein-containing system in which high concentrations of proline and/or betaine are present, is better ;protected' against the biologically unfavorable consequences of dehydration-induced thermodynamic perturbation.

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.

Correlation between the Lipid Composition and the Responsiveness of Avena sativa Stem Segments to Gibberellic Acid.

The lipid composition of Avena sativa stem segments was manipulated using BASF 13-338 (formerly Sandoz 9785) and growth temperature, in order to establish whether there were correlations between responsiveness of the tissue to gibberellic acid (GA(3)) and the presence, before hormone treatment, of specific lipid components. High correlations were obtained between GA(3)-induced growth and total phospholipid, individual phospholipids, and fatty acids (except for linolenic acid), total saturated fatty acids, stigmasterol content, and the unsaturated/saturated fatty acid ratio. It was concluded that, although the lipid composition, and particularly the total saturated fatty acid content, seem to be important contributory determinants of the GA(3)-induced growth response in this system, they may not be obligatory prerequisites, nor the only endogenous factors capable of influencing the response. However, the results are consistent with the hypothesis that membranes are involved in the hormonal mechanism and/or very early stages of the mode of GA(3) action in this tissue.

The Influence of Gibberellic Acid and Temperature on the Growth Rate of Avena sativa Stem Segments.

A micro-growth measuring technique was used to determine the growth response of stem segments of Avena sativa cv. Avon to a variety of gibberellic acid (GA(3)) concentrations over a range of incubation temperatures. Growth rate varied with GA(3) concentration, the temperature at which the rate was measured, and the growth temperature of the plants prior to excision of the segments. The curves relating segment extension rates to temperature were affected by GA(3) such that the linear portion of the curve was shifted to higher rates as GA(3) concentration was increased. The results seem to be analogous to the GA(3)-induced shifts of thermally induced phase transitions in glucose leakage from liposomes, observed earlier (Wood, Paleg 1974 Aust J Plant Physiol 1: 31-40).

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.

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.

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.

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.

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.