Effects of 2,4-dinitrophenol on membrane lipids of roots

Previous work has shown that the undissociated form of 2,4-dinitrophenol (DNP) increases the permeability of barley (Hordeum vulgare var. trebi) roots to ions. The present studies were undertaken to determine whether the effects of undissociated DNP were directly on membrane lipids. Relative amounts of the principal fatty acids from the lipids of barley root membranes were assayed as a function of DNP concentration, pH, and time of treatment under conditions similar to the previous studies of DNP effects on permeability. Undissociated DNP increases the proportions of palmitic and oleic acids and decreases linoleic and linolenic acids with no changes in the amounts of total fatty acids. The effects are immediate, as are the effects on permeability. Only the undissociated DNP is effective. Anionic DNP has no effect, although it is the major species taken up by the roots both at pH 5 and pH 7. DNP has no effect on respiration at either pH, indicating that undissociated DNP effects are on the membranes and not a general metabolic effect. The close parallelism between the effects of DNP on the composition of membrane lipids and on permeability suggests that the increase in permeability produced by undissociated DNP is due to a direct effect on the root membranes.     

Effects of organic acids on ion uptake and retention in barley roots

Effects of several organic acids on ion uptake and retention and on respiration in barley roots having low and high KCl contents were assayed by measurements of K⁺, Na⁺, Ca²⁺, Cl⁻, and oxygen uptake. Organic acids with high pK_a values increase the permeability of roots to ions and decrease respiration when present in sufficient concentrations at pH 5 but have no inhibitory effects at pH 7. Absence of respiratory inhibition in short times and at lower organic acid concentrations, under conditions that immediately produce a permeability increase, indicate that the permeability change is not a result of respiratory inhibition. Effects of formate, acetate, propionate, and glutarate are attributed to entry of undissociated acid molecules into the effective membranes. Lack of a permeability increase with succinate, which has lower distribution coefficients to lipid solvents than do the aliphatic acids, can be explained by failure of sufficient amounts of the hydrophilic succinic acid molecules to penetrate the membranes involved. These experiments suggest that undissociated acid in root membranes can increase permeability of the roots.     

Differences between Effects of Undissociated and Anionic 2,4-Dinitrophenol on Permeability of Barley Roots

Effects of 2,4-dinitrophenol (DNP) and several other substituted phenols on permeability of barley roots (Hordeum vulgare var. Trebi) to ions were assayed as a function of pH and phenol concentration. Solutions containing 0.1 micromolar undissociated DNP increase the permeability of barley root cells to small ions such as K⁺, Na⁺, Ca²⁺, and Cl⁻ with no inhibition of respiration. Undissociated forms of the other phenols increase permeability also, but they are less effective than DNP. Only the undissociated DNP is effective. Anionic DNP does not increase permeability or inhibit ion uptake, although it is the major species accumulated by the roots, both at pH 5 and pH 7. At pH 7, in contrast to pH 5, 10 micromolar DNP has no effect on ion permeability of barley roots yet it uncouples oxidative phosphorylation of barley root mitochondria. This indicates that the all too common use of DNP as a test for active transport or involvement of ATP synthesis can be misleading.     

Use of P NMR to Assess Effects of DNP on ATP Levels in Vivo in Barley Roots

Previous work has shown that undissociated 2,4-dinitrophenol (DNP) both increases the permeability of roots to ions and alters the membrane lipids of barley roots. Anionic DNP is the main entrant form but has no effect on permeability or on the membrane lipids. The amount of anionic DNP taken up by the roots is sufficient, that were it in free solution in the cytoplasm, the DNP would uncouple oxidative phosphorylation, and thereby inhibit ATP synthesis. The present work was undertaken to assess whether DNP alters ATP levels when it is taken up by barley roots. ³¹P nuclear magnetic resonance spectra were used to monitor, in vivo, levels of ATP, cytoplasmic phosphate, vacuolar phosphate, and other phosphate compounds in barley roots in the presence of 10 micromolar DNP at pH 5 and pH 7. The spectra indicate that no change in the level of ATP or the cytoplasmic pH occurred in the roots in the presence of DNP for as long as 20 hours. Thus, the effects of undissociated DNP are effects directly on the root membranes and do not involve inhibition of ATP synthesis. Furthermore, the results explain why anionic DNP has no effect on ion uptake and accumulation.     

Adaptation of H+-pumping and plasma membrane H+ ATPase activity in proteoid roots of white lupin under phosphate deficiency

White lupin (Lupinus albus) is able to adapt to phosphorus deficiency by producing proteoid roots that release a huge amount of organic acids, resulting in mobilization of sparingly soluble soil phosphate in rhizosphere. The mechanisms responsible for the release of organic acids by proteoid root cells, especially the trans-membrane transport processes, have not been elucidated. Because of high cytosolic pH, the release of undissociated organic acids is not probable. In the present study, we focused on H+ export by plasma membrane H+ ATPase in active proteoid roots. In vivo, rhizosphere acidification of active proteoid roots was vanadate sensitive. Plasma membranes were isolated from proteoid roots and lateral roots from P-deficient and -sufficient plants. In vitro, in comparison with two types of lateral roots and proteoid roots of P-sufficient plants, the following increase of the various parameters was induced in active proteoid roots of P-deficient plants: (a) hydrolytic ATPase activity, (b) Vmax and Km, (c) H+ ATPase enzyme concentration of plasma membrane, (d) H+-pumping activity, (e) pH gradient across the membrane of plasmalemma vesicles, and (f) passive H+ permeability of plasma membrane. In addition, lower vanadate sensitivity and more acidic pH optimum were determined for plasma membrane ATPase of active proteoid roots. Our data support the hypothesis that in active proteoid root cells, H+ and organic anions are exported separately, and that modification of plasma membrane H+ ATPase is essential for enhanced rhizosphere acidification by active proteoid roots.     

Heat resistance of Paenibacillus polymyxa in relation to pH and acidulants

The efficacy of different organic acids in decreasing the heat resistance of Paenibacillus polymyxa spores was assessed. The relationship between concentration of the undissociated form of different organic acids and decrease in heat resistance was also investigated. The heat resistance of P. polymyxa spores was tested in distilled water at 85, 90 and 95 degrees C, at pH4 and in the presence of 50, 100 and 200 mmol l(-1) of the undissociated form of lactic, citric or acetic acid and sodium citrate or acetate. The undissociated form of organic acids was responsible for increasing the heat sensitivity of spores. The most effective acid was lactic acid. The D values of the spores decreased rapidly (between 74 and 43%) in the presence of 50 mmol l(-1) of the undissociated form of organic acid, and increasing concentrations of these forms affected the heat resistance of spores less than proportionally. The heat resistance of the spores in milk was approximately threefold lower than in distilled water. This work has shown that the undissociated fraction of organic acids increases, albeit non-linearly, the sensitivity of spores to heat, even in complex substrates such as milk. By knowing the amount of organic acids added to a given substrate, their dissociation constants and the final pH, it could be possible to estimate the concentration of undissociated forms and the corresponding increase in lethality of heat treatments. This would help the food industry to maximize the lethality achieved by heat processes and/or safely reduce the heat treatments already in use.     

Phragmites die-back: toxic effects of propionic, butyric and caproic acids in relation to pH

Symptoms which are associated with die-back in Phragmites : growth inhibition, root and bud death, premature shoot senescence, blocked aeration and vascular systems, especially in rhizomes and roots, and abnormal surface and internal cell-wall lignification and suberization of roots were induced by each of three of the lower volatile organic acids, propionic, butyric and caproic. These acids were applied in nutrient media in concentrations similar to those previously associated with die-back sites and/or in sediments containing rotting rhizomes and roots of the plant. At concentrations of 1.4 and 0.56 mM, respectively, butyric and caproic acids were each found to be highly toxic at pH 4.5, but relatively innocuous at pH 6. Propionic acid, applied at a much higher concentration of 10.4 mM, was highly toxic at both pH 4.5 and 6. The results support previous findings that the undissociated forms of the organic acids are the more toxic. Rhizomes and roots, rotting in water or waterlogged sand, released cocktails of acids and produced pH in the range 4.8–5.4. Phragmites seedlings planted in these media died within 12 h. Overall, the results support the theory that die-back in Phragmites can be induced and/or perpetuated by organic acids released from the decaying underground parts of the plant or other sources of organic matter.     

Root exudation and rhizoplane bacterial abundance of barley (Hordeum vulgare L.) in relation to nitrogen fertilization and root growth

The abundance of bacteria in the rhizoplane of barley varieties was investigated at different soil nitrogen levels. Increased amendments of nitrogen resulted in higher bacterial numbers in the rhizoplane of barley seedlings of different varieties. A negative correlation was found between nitrogen level in the soil and the growth rate of the seedling roots. The effect of nitrogen on the bacterial abundances could be indirect through changed root growth and thereby changed exudation. The exudation of soluble organic carbon componds from barley seedling roots were measured in hydroponic culture. The effect of natural variation in root growth rate and of different concentrations of nitrogen in the nutrient solution was investigated. The amount of exudates consituted 2–66% of the dry weight increase in root biomass, depending on the root growth. Slower growing roots released considerably more organic carbon per unit root weight than faster growing roots. The variation in root exudation appeared to be mainly explained by differences in root growth, rather than of the nitrogen concentration in the nutrient solution. A significantly higher exudation rate was found during day time compared to night.     

Isolation of biochemical mutants using haploid mesophyll protosplasts of Hyoscyamus muticus

The question of whether membrane expansion, which is caused by anesthetics in animal systems, alters the lipid composition of plant cell membranes was investigated. We have measured the effects of several anesthetics on the relative amounts of the principal fatty acids from the polar lipids of barley (Hordeum vulgare L.) root membranes. Procaine, dibucaine, tetracaine, chloroform and, to a lesser degree, methanol increased the proportions of palmitic, stearic and oleic acids and decreased the proportions of linoleic and linolenic acids. Ethanol had no significant effect. Total amounts of the fatty acids from the polar lipids of roots in procaine solution decreased markedly so that all of the acids decreased in amount. The anesthetic was effective as soon as the roots were introduced to the solution and the changes progressed at constant rates for 6 h. Only the polar membrane lipids were altered; other lipids were not affected. Increased hydrostatic pressure of about 1.0 MPa largely prevented the anesthetic effects, including the decrease in the total amounts of the fatty acids. Hydrostatic pressure as high as 2 MPa had no effect per se on the membrane lipid composition. These results indicate that anesthetics cause expansion of the root membranes which results in the lipid changes. That a compositional change in the membrane lipids involves a conformational change such as expansion is an indication of the nature of the link between changes in the membrane lipids and changes in function of areas where hydrophilic ions permeate.Abbreviations 16:0 palmitic acid - 18:0 stearic acid - 18:1 oleic acid - 18:2 linoleic acid - 18:3 linolenic acid     

The Release of Nitrite from Barley Roots in Response to Metabolic Inhibitors, Uncoupling Agents, and Anoxia

When excised sterile barley roots, from plants which had beengrown in the presence of nitrate, were placed under low oxygentensions, nitrite was released into the external solution. Themaximum leakage of nitrite occurred under completely anaerobicconditions. Nitrite was also released from barley roots underaerobic conditions when uncouplers of oxidative phosphorylation(DNP, CCCP¹, pentachlorophenol) or certain simple organic acidswere supplied. Inhibitors of the Krebs cycle, or of respiratoryelectron transport, were much less effective in causing theloss of nitrite, possibly because these compounds did not ingeneral inhibit root respiration severely. Nitrite release, in response to any of the above treatments,was accompanied by an accumulation of nitrite within the tissue.It was concluded that an increase in membrane permeability,and a decrease in ATP synthesis, were contributory causes ofthis phenomenon, although neither could explain the experimentalobservations completely. There was however no evidence thatthe pentose phosphate pathway, which is regarded as the sourceof reducing power for nitrite reduction, was inhibited underconditions which favoured nitrite release.     

Changes of membrane permeability due to extensive cholesterol depletion in mammalian erythrocytes

55% of the total membrane cholesterol could be removed from porcine, bovine and human erythrocytes by incubating the cells in suspensions of lecithin liposomes. Up to 30% depletion, membrane permeability remained unaltered; more extensive depletion induced a marked increase of the transfer rates of nonelectrolytes and of organic acids penetrating by nonionic diffusion. This biphasic response of permeability to cholesterol depletion, which has not been observed in artificial lipid membranes, may be related to the heterogeneity of the erythrocyte membrane lipids or to a pool of cholesterol not interacting with the phospholipids.     

Loss of organic acids, amino acids, k, and cl from barley roots treated anaerobically and with metabolic inhibitors

Excised roots of barley (Hordeum vulgare, var. Campana) lost organic acids, amino acids, K⁺, and Cl⁻ within 15 minutes after initiation of anaerobic treatment or treatment with NaCN and 2,4-dinitrophenol. Initial loss of organic acids when roots were placed under N₂ is attributed to a decarboxylation reaction, possibly catalyzed by phosphoenolpyruvate carboxykinase. Organic and amino acids began to leak from the roots to the bathing medium after 1 to 2 hours under N₂, indicating injury to cell membranes. During the first hour of anaerobic treatment, K⁺ loss from low-salt roots was equivalent to organic acid loss. Potassium loss from roots containing high levels of KCl was approximately equal to organic acid plus amino acid loss; and Cl⁻ loss was approximately equal to amino acid loss. It is postulated that, within cells, organic acids may electrostatically bind an equivalent quantity of cations and that amino acids may bind an equivalent quantity of both cations and anions.     

Quantitative and qualitative effects of phosphorus on extracts and exudates of sudangrass roots in relation to vesicular-arbuscular mycorrhiza formation

A comparison was made of water-soluble root exudates and extracts of Sorghum vulgare Pers. grown under two levels of P nutrition. An increase in P nutrition significantly decreased the concentration of carbohydrates, carboxylic acids, and amino acids in exudates, and decreased the concentration of carboxylic acids in extracts. Higher P did not affect the relative proportions of specific carboxylic acids and had little effect on proportions of specific amino acids in both extracts and exudates. Phosphorus amendment resulted in an increase in the relative proportion of arabinose and a decrease in the proportion of fructose in exudates, but did not have a large effect on the proportion of individual sugars in extracts. The proportions of specific carbohydrates, carboxylic acids, and amino acids varied between exudates and extracts. Therefore, the quantity and composition of root extracts may not be a reliable predictor of the availability of substrate for symbiotic vesicular-arbuscular mycorrhizal fungi. Comparisons of the rate of leakage of compounds from roots with the growth rate of vesicular-arbuscular mycorrhizal fungi suggest that the fungus must either be capable of using a variety of organic substrates for growth, or be capable of inducing a much higher rate of movement of specific organic compounds across root cell membranes than occurs through passive exudation as measured in this study.     

Acid phosphatase activity in phosphorus-deficient white lupin roots

White lupin ( Lupinus albus L.) develops proteoid roots when grown in phosphorus (P)-deficient conditions. These short, lateral, densely clustered roots are adapted to increase P availability. Previous studies from our laboratory have shown proteoid roots have higher rates of non-photosynthetic carbon fixation than normal roots and altered metabolism to support organic acid exudation, which serves to solubilize P in the rhizosphere. The present work indicates that proteoid roots possess additional adaptations for increasing P availability and possibly for conserving P in the plant. Roots from P-deficient (–P) plants had significantly greater acid phosphatase activity in both root extracts and root exudates than comparable samples from P-sufficient (+P) plants beginning 10 d after emergence. The increase in activity in –P plants was most pronounced in the proteoid regions. In contrast, no induction of phytase activity was found in –P plants compared to +P plants. The number of proteoid roots present was not affected by the source of phosphorus supplied, whether organic or inorganic forms. Adding molybdate to the roots increased the number of proteoid roots in plants supplied with organic P, but not inorganic P. Increased acid phosphatase activity was detected in root exudates in the presence of organic P sources. Native-polyacrylamide gel electrophoresis demonstrated that under P-deficient conditions, a unique isoform of acid phosphatase was induced between 10 and 12 d after emergence. This isoform was found not only within the root, but it comprised the major form exuded from proteoid roots of –P plants. The fact that exudation of proteoid-root-specific acid phosphatase coincides with proteoid root development and increased exudation of organic acids indicates that white lupin has several coordinated adaptive strategies to P-deficient conditions.     

Changes in Chloroplast Membrane Lipids during Adaptation of Barley to Extreme Salinity

During adaptation of barley (Hordeum vulgare L.) seedlings to extremely high concentrations of sodium chloride in the root space, the content of galactolipids of chloroplast membranes decreased considerably. Alterations in membrane lipids were due to the high concentration of ions rather than to the increase in the water potential. Sodium chloride was accumulated in the leaf cells and affected lipid-synthesizing enzymes such as galactosyl transferase and acylase which are attached to the chloroplast envelope. The return of salt-adapted barley seedlings to a nutrient solution with low salt concentration resulted in a reversal of the observed changes. It is suggested that the decrease in content of galactolipids in biomembranes is one of the factors causing increased salt resistance in barley plants which are adapted to extreme salinity.     

不同氮素形态培养下荞麦叶片中草酸积累的变化

用1/5浓度Hoagland(pH6.0)营养液培养荞麦幼苗3d后,取其中一部分继续用此营养液(硝态氮);另一部分用硫酸氨和氯化钙取代硝态氮(氨态氮)的营养液,均培养至荞麦第一片真叶完全展开。结果表明,以氨态氮为唯一氮源培养荞麦时,植株叶片中草酸含量显著下降。进一步研究表明,氨态氮培养下荞麦根中及根分泌草酸的速率也显著下降,结果排除了叶片中草酸含量的下降是由于叶片中草酸向其根系转运或是因为根分泌草酸速率的差异造成的,而可能与其草酸代谢改变有关。氨态氮培养下叶片中与草酸代谢相关的有机酸含量以及相关酶活性也显著下降,这可能意味着荞麦叶片草酸形成积累可能与相关有机酸代谢有关。     

锌营养状况对小麦根细胞膜透性的影响

小麦缺锌不仅导致根系K~ 和NO_3~-泌出量增加,而且低分子量有机化合物如氨基酸、糖类化合物和酚类化合物的泌出量也明显提高。重新供锌(ZnSO_4)12h后,根系K~ 、NO_3~-、氨基酸和碳水化合物的泌出量迅速减少,随着时间的延长,泌出量接近对照水平。结果说明锌对根细胞膜结构的稳定性及膜功能的完整性是必不可少的。     

Short-term aluminium-induced changes in barley root tips

The short-term exposure of barley roots to low Al concentration caused significant root growth inhibition and radial swelling of roots. During Al treatment, the radial expansion of root cells occurred in root tissues representing elongation zone and meristem. Both low pH and Al treatments caused significant disruption of cell membranes in swollen roots. In contrast to Evans blue uptake callose formation was observed only at higher Al concentrations and was detected in both swollen and adjacent root areas. Similarly to Al, exogenous short-term application of indole-3-acetic acid, polar transport inhibitor triiodobenzoic acid, ethylene precursor 1-aminocyclopropane-1-carboxylic acid or H₂O₂ evoked root growth inhibition and radial cell expansion in barley root tip too.     

Root exudates of plants

Summary The composition of the root exudates of barley and wheat in the initial growth phases was investigated; amino acids, organic acids, sugars and certain aromatic compounds could be identified. A knowledge of the composition of root exudates is important from the standpoint of the interaction between the plants and the micro-organisms in the rhizosphere. Some aspects of the rhizosphere effect connected with the present work are discussed.     

Lipids from Bean, Barley and Sugar Beet in Relation to Salt Resistance

A comparison was made between the lipid and fatty acid composition of the salt-sensitive bean (Phaseolus vulgaris L. cv. Saxa), the less salt-sensitive barley (Hordeum vulgaris L. cv. Wisa) and the salt-tolerant sugar beet (Beta vulgaris L. cv. Kawemono). Sugar beet roots showed a higher content of sterol components and sulfolipid as compared with bean and barley roots. The lipids of sugar beet roots contained more linoleic acid and less linolenic acid than those of bean and barley roots. For barley and sugar beet roots a higher amount of extra-long chain fatty acids was observed than for bean roots. It was concluded that differences in membrane structure are correlated with differences in membrane permeability to sodium and chloride and in salt-resistance of the studied species.