On the Relationship between Extracellular pH and the Growth of Excised Pea Stem Segments

Studies with stem segments of peas (Pisum sativum L. var. Alaska) suggest that the pH of the medium bathing elongating tissue does not always reflect intramural (cell wall) conditions or that pH is not a controlling factor in elongation. Peeled, green segments, and peeled or nonpeeled etiolated segments appear to regulate the pH of their bathing medium causing it to become acidified with or without the addition of auxin. The growth rates of segments are greatest during a period before acidification is evident and slow during the time in which the medium becomes acidified. We cannot reproduce the dramatic auxin-induced pH shifts reported in the literature because the control segments are becoming more acid also; but there is some evidence that acidification may occur in response to auxin treatments. K⁺ additions mimic the acidifying tendency of auxin but are without growth-promoting effect. Emergent growth (an extremely rapid burst of growth following anaerobic treatments) is not accompanied by a drop in pH of the bathing medium. Proper aeration of the bathing medium in extracellular pH studies is crucial and may explain differences between our results and other published accounts. The data suggest that the techniques used for most extracellular pH studies may not very closely approximate in vivo conditions or properly reflect intramural H⁺ concentration fluxes.     

Effect of cell-wall-digesting enzymes on physiological state and competence of maize coleoptile cells

Summary Protoplasts are frequently isolated from maize coleoptiles with cell-wall-degrading enzymes such as pectolyase (PEC), mazerozyme, and cellulase. Incubation of coleoptiles with these enzymes caused rapid depolarizations of the membrane voltage (V _M ). The depolarizing effect of 0.5% (w/v) mazerozyme or 1.5% (w/v) cellulase was unaffected by denaturation of the enzymes. In the case of pectolyase (0.1%, w/v), however, the active enzyme was significantly more potent than the denaturated enzyme in depolarizing coleoptile cells. Exposure to 0.1% active PEC but not to inactive PEC also caused an oxidative burst in coleoptiles and enhanced K⁺ efflux. Together this suggests that pectic breakdown products of the cell wall act as signal for wounding. Typically addition of 10 M 1-naphthylene acetic acid (NAA) to coleoptiles causes a transient depolarization followed by a slow hyperpolarization of V _M . However, in the presence of PEC, V _M only depolarized in NAA. After PEC-treated coleoptiles were washed free of the enzyme, NAA caused only small fluctuations of V _M . A similarly small V _M response to NAA appeared in coleoptiles pretreated with heatdenaturated supernatant (SUP) from a protoplast isolation buffer, the latter suspected to contain the PEC-generated wounding signal. Comparable pretreatment of coleoptiles with PEC or SUP had no significant effect on the spontaneous and NAA-evoked acidification of the incubation medium. Pretreatment with SUP also had no significant effect on the NAA-stimulated elongation of coleoptile segment. Hence, PEC treatment of coleoptile tissue affects the membrane transport properties of the cells. This effect is partly maintained after removal of the enzyme from the incubation medium, an effect not significant for NAA-generated acidification and cell elongation.Abbreviations V _M membrane voltage - V_red redox voltage - PEC pectolyase - SUP supernatant from cell wall digestion - NAA 1-naphthylene acetic acid     

Effect of thiosulphinates contained in garlic extract on growth, proton fluxes and membrane potential in maize (Zea mays L.) coleoptile segments

The effect of thiosulphinates contained in garlic extract (GE) on endogenous growth, growth in the presence of either indoleacetic acid (IAA) or fusicoccin (FC), and proton extrusion in maize coleoptile segments were studied. In addition, membrane potential changes at some GE dilutions and the protective effect of dithiothreitol (DTT) against GE toxicity were also determined. It was found that GE at almost all dilutions studied, when added to the incubation medium inhibited endogenous growth as well as growth in the presence of either IAA or FC. Simultaneous measurements of growth and external pH indicated that the administration of GE resulted in a complex change in the pH of the external medium; after an initial transient acidification, pH increased and reached the maximal value followed by a gradual decrease of medium pH. When IAA or FC was added after preincubation of the segments in the presence of GE the changes in medium pH were not significantly different from these obtained with GE only. If the coleoptile segments were first preincubated with GE and subsequently GE was removed, the addition of IAA induced strong growth and medium acidification. Dithiothreitol added together with GE neutralized the toxic effect of GE on growth of coleoptile segments incubated in the presence of IAA. The addition of GE to the control medium caused a depolarization of the membrane potential, the value of witch depended on GE dilution. These results indicate that the toxic effect of GE on growth of plant cells might be caused by disruption of the catalytic function of the plasma membrane H⁺-ATPase on formation of the disulfide bonds.     

Cellular responses to naphthoquinones: juglone as a case study

The effects of juglone (JG) on the endogenous growth, growth in the presence of either indoleacetic acid (IAA) or fusicoccin (FC) and on proton extrusion were studied in maize coleoptile segments. In addition, membrane potential changes were also determined at chosen JG concentrations. It was found that JG, when added to the incubation medium, inhibited endogenous growth as well as growth in the presence of either IAA or FC. Simultaneous measurements of growth and external pH indicated that inhibition of either IAA-induced growth or proton extrusion by JG was a linear function of JG concentration. Addition of JG to the control medium caused depolarization of the membrane potential (E_m), value of which was dependent on JG concentration and time after its administration. Hyperpolarization of E_m induced by IAA was suppressed in the presence of JG. It was also found that for coleoptile segments initially preincubated with JG, although subsequently removed, addition of IAA was not effective in the stimulation of growth and medium acidification. Taken together, these results suggest that the mechanism by which JG inhibits the IAA-induced growth of maize coleoptile segments involves inhibition of PM H⁺-ATPase activity.     

Totipotency of coleoptile tissue in indica rice (Oryza sativa L. cv. ch 1039)

Embryogenic and non-embryogenic calluses were induced from 3,4,5 and 7d old coleoptile segments of indica rice (Oryza sativa L. cv. CH 1039). Compact, globular, yellow and creamy embryogenic and white friable non-embryogenic callus arose from the cut end and entire length of the coleoptile segments. Murashige and Skoog's (MS) medium supplemented with 2.5mg/1 2,4-D was used as callus induction medium. Plant regeneration from coleoptile segments occurred with the transfer of embryogenic callus to MS basal medium supplemented with 2.0mg/1 BAP and 0.5mg/1 NAA in combination. Average number of regenerated plants from one coleoptile ranged from9.1 to 14.0.Four day old coleoptiles showed the highest frequency of plant regeneration.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - MS Murashige and Skoog (1962) - NAA 1-naphthalene acetic acid     

In vivo evidence for the involvement of phospholipase A and protein kinase in the signal transduction pathway for auxin-induced corn coleoptile elongation

Auxin-induced elongation of com coleoptiles is accompanied by cell wall acidification, which depends upon H⁺-pump activity. We tested the hypothesis that phospholipase A and a protein kinase are involved in the pathway of auxin signal transduction leading to H⁺ secretion, and elongation of corn coleoptiles. Initially, the pH of the bath solution at 50–100 μm from the surface of a coleoptile segment (pH_o) ranged between 4.8 and 6.6 when measured with an H⁺-sensitive microelectrode. Twenty or 50 μM lysophosphatidylcholine, 50 μM linolenic acid or 50 μM arachidonic acid induced a decline in pH_o by 0.3 to 2.1 units. The effect was blocked by 1 mM vanadate, suggesting that lysophosphatidylcholine or linolenic acid induced acidification of the apoplast by activating the H⁺-pump. Lysophosphatidylcholine and linolenic acid also accelerated the elongation rate of the coleoptiles. While linolenic acid and arachidonic acid, highly unsaturated fatty acids, promoted pH_o decrease and coleoptile elongation, linoleic acid, oleic acid, and stearic acid, fatty acids with a lesser extent of unsaturation, had no such effects. The effects of lysophosphatidylcholine, linolenic acid, and arachidonic acid on H⁺ secretion were not additive to that of indoleacetic acid (IAA), suggesting that lysophospholipids, fatty acids and auxin use similar pathways for the activation of the H⁺-pump. The phospholipase A₂ inhibitors, aristolochic acid and manoalide, inhibited the IAA-induced pH_o decrease and coleoptile elongation. The general protein kinase inhibitors, H-7 or staurosporine, blocked the IAA- or lysophosphatidylcholine-induced decrease in pH_o. H-7 also inhibited the coleoptile elongation induced by IAA or lysophosphatidylcholine. These results support the hypothesis that phospholipase A is activated by auxin, and that the products of the enzyme, lysophospholipids and fatty acids, induce acidification of the apoplast by activating the H⁺-pump through a mechanism involving a protein kinase, which in turn promotes com coleoptile elongation.     

Photoreversible calcium fluxes induced by phytochrome in oat coleoptile cells

The chromometallic dye murexide was used to measure photoreversible Ca fluxes in apical tips of etiolated oat coleoptiles and in suspension cultures of protoplasts derived from the coleoptile segments. Phytochrome presence in the protoplasts was indicated by a repeatably photoreversible ΔA(725 - 800 nm) of >0.001 A centimeters⁻¹, recorded on a dual wavelength spectrophotometer. Concentrations of Ca in the solution bathing the cells were observed to change photoreversibly, red irradiation inducing an increase in the medium Ca concentration and subsequent farred irradiation inducing a decrease down to near dark control levels. These changes could be measured in media with or without exogenously added Ca. Protoplasts from green primary leaves of oat, which had no spectro-photometrically detectable phytochrome, showed no photoreversible Ca fluxes when measured by the same method. These data imply that red light induces an efflux of Ca from phytochrome-containing cells and that far red light can reverse this change by promoting a Ca reentry into these cells.     

Acetate production byClostridium thermoaceticum in corn steep liquor media

A low-cost nutrient medium based on corn steep liquor (CSL) was developed for the production of acetates byClostridium thermoaceticum. Pre-treatment of CSL with dolime and vitamin supplementation increased the rate of acetate production. Adding excess nutrients in a fed-batch mode minimized by-product formation and increased final acetate concentration from 19 g L^–1 to 40 g L^–1 acetic acid. High yields of acetic acid (0.95 g g^–1 glucose in fed-batch mode) was probably due to the conversion of the lactic acid in CSL into acetic acid by the organism.     

Red light and auxin effects on rubidium uptake by oat coleoptile and pea epicotyl segments

Apical segments of etiolated oat (Avena sativa L. cv. Victory) coleoptiles showed enhanced uptake of [⁸⁶Rb⁺] when tested 30 minutes after a 5-minute red irradiation. The response was partly reversible by far red light. Uptake was sensitive to carbonyl cyanide m-chlorophenyl hydrazone, but not to isotonic mannitol. Indoleacetic acid (10⁻⁷ molar) caused a very pronounced and rapid stimulation of uptake. Basal coleoptile segments also exhibited a red light-enhanced uptake, but not an effect of red light on changes in the pH of the medium. The [⁸⁶Rb⁺] uptake of third internode segments from etiolated peas (Pisum sativum L. cv. Alaska) was not affected by either red light or auxin. This tissue also showed no red light effect on acidification of the medium. It is concluded that alteration of [⁸⁶Rb⁺] flux is not a general feature of phytochrome action.     

Effect of temperature and sorbitol in improving the solubility of carboxylesterases protein CpCE-1 from Cydia pomonella and biochemical characterization

Carboxylesterases (CEs) are enzymes responsible for the detoxification of insecticides in insects. In the Cydia pomonella, CEs are involved in synthetic pyrethroid, neonicotinoid, carbamate, and organophosphate detoxification. However, functional overexpression of CEs proteins in Escherichia coli systems often results in insoluble proteins. In this study, we expressed the fusion protein CpCE-1 in E. coli BL21 (DE3). This recombinant protein was overexpressed as inclusion bodies at 37 °C whereas it produced a higher percentage of soluble protein at lower growth temperatures. Production of soluble proteins and enzyme activity increased in the presence of sorbitol in the growth medium. The fusion protein was purified from the lysate supernatant using a Ni²⁺-NTA agarose gel column. The enzyme exhibited a higher affinity and substrate specificity for α-naphthyl acetate (α-NA), with k _cat/K _m of 100 s^?1 μM^?1 for α-NA, and the value is 29.78 s^?1 μM^?1 for β-naphthyl acetate. The V _max and K _m were also determined to be 12.9 μmol/min/mg protein and 13.4 μM using substrate α-NA. The optimum pH was 7.0 and temperature was 25 °C. An enzyme inhibition assay shows that PMSF and DEPC strongly inhibit the enzyme activity, while the metal ions Cu²⁺ and Mg²⁺ significantly activated the activity. More importantly, cypermethrin, methomyl, and acephate were found to suppress enzyme activity. The data demonstrated here provide information for heterologous expression of soluble protein and further study on insecticide metabolism in C. pomonella in vitro. This is the first report of the characterization of CEs protein from C. pomonella.     

Influence of Phenolic Acids on Ion Uptake: IV. Depolarization of Membrane Potentials

The membrane potentials of aged, excised barley (Hordeum vulgare L.) root cells were rapidly depolarized by the addition of salicylic acid (o-hydroxybenzoic acid) to the buffered medium bathing root segments. Initial values for membrane potentials were restored very slowly (within 100 minutes) by replacing the phenolic solution by phenolic-free buffer. Several other naturally occurring benzoic and cinnamic acids depolarized cell membrane potentials. The cinnamic acids consistently caused a greater depolarization than the correspondingly substituted benzoic acids. A strong positive correlation was found between the depolarization values (ΔE) for the benzoic acids and their lipid solubilities. This study supports the hypothesis that the inhibition of ion uptake brought about by naturally occurring phenolic acids is caused by a generalized increase in membrane permeability to inorganic ions.     

Evidence for Na-H Exchange in the Rabbit Pancreas

WE have found that pancreatic juice secretion can be maintained at a high rate even when all exogenous HCO₃ is replaced by another weak acid anion such as acetate. When acetate is secreted by the rabbit pancreas in vitro, juice flows at rates of 30–90% of control¹ and acetate reaches a concentration up to three times that in the ambient bathing solution. HCO₃ is not essential for juice production and so these data suggest that active transport of H or OH ions is responsible for normal HCO₃ secretion. We have therefore examined the effect of alterations of ambient pH on cell pH. Our data indicate the existence of an Na-H exchange mechanism at the serosal (bath-facing) membrane.     

Can Lateral Redistribution of Auxin Account for Phototropism of Maize Coleoptiles?

Elongation growth of intact, red-light grown maize (Zea mays L.) coleoptiles was studied by applying a small spot of an indole acetic acid (IAA)-lanolin mixture to the coleoptile tip. We report that: (a) endogenous auxin is limiting for growth, (b) an approximately linear relation holds between auxin concentration and growth rate over a range which spans those rates occurring in phototropism, and (c) an auxin gradient established at the coleoptile tip is well sustained during its basipetal transport. We argue that the growth differential underlying coleoptile phototropism (first-positive curvature) can be explained by redistribution of auxin at the coleoptile tip.     

Beneficial Effect of Acetic Acid on the Xylose Utilization and Bacterial Cellulose Production by Gluconacetobacter xylinus

In this work, acetic acid was found as one promising substrate to improve xylose utilization by Gluconacetobacter xylinus CH001. Also, with the help of adding acetic acid into medium, the bacterial cellulose (BC) production by G. xylinus was increased significantly. In the medium containing 3 g l⁻¹ acetic acid, the optimal xylose concentration for BC production was 20 g l⁻¹. In the medium containing 20 g l⁻¹ xylose, the xylose utilization and BC production by G. xylinus were stimulated by acetic acid within certain concentration. The highest BC yield (1.35 ± 0.06 g l⁻¹) was obtained in the medium containing 20 g l⁻¹ xylose and 3 g l⁻¹ acetic acid after 14 days. This value was 6.17-fold higher than the yield (0.21 ± 0.01 g l⁻¹) in the medium only containing 20 g l⁻¹ xylose. The results analyzed by FE-SEM, FTIR, and XRD showed that acetic acid affected little on the microscopic morphology and physicochemical characteristics of BC. Base on the phenomenon observed, lignocellulosic acid hydrolysates (xylose and acetic acid are main carbon sources present in it) could be considered as one potential substrate for BC production.     

Plant Growth Substances Produced by Azospirillum brasilense and Their Effect on the Growth of Pearl Millet (Pennisetum americanum L.)

Azospirillum brasilense, a nitrogen-fixing bacterium found in the rhizosphere of various grass species, was investigated to establish the effect on plant growth of growth substances produced by the bacteria. Thin-layer chromatography, high-pressure liquid chromatography, and bioassay were used to separate and identify plant growth substances produced by the bacteria in liquid culture. Indole acetic acid and indole lactic acid were produced by A. brasilense from tryptophan. Indole acetic acid production increased with increasing tryptophan concentration from 1 to 100 μg/ml. Indole acetic acid concentration also increased with the age of the culture until bacteria reached the stationary phase. Shaking favored the production of indole acetic acid, especially in a medium containing nitrogen. A small but biologically significant amount of gibberellin was detected in the culture medium. Also at least three cytokinin-like substances, equivalent to about 0.001 μg of kinetin per ml, were present. The morphology of pearl millet roots changed when plants in solution culture were inoculated. The number of lateral roots was increased, and all lateral roots were densely covered with root hairs. Experiments with pure plant hormones showed that gibberellin causes increased production of lateral roots. Cytokinin stimulated root hair formation, but reduced lateral root production and elongation of the main root. Combinations of indole acetic acid, gibberellin, and kinetin produced changes in root morphology of pearl millet similar to those produced by inoculation with A. brasilense.     

The use of mammalian cultured cells loaded with a fluorescent dye shows specific membrane penetration of undissociated acetic acid

Acetic acid induces unique physiological responses in mammalian cells. Our previous study found that fura-2-loaded human embryonic kidney (HEK) 293T cells showed a robust intracellular fluorescence response immediately after stimulation with acetic acid, and no such response in the case of citric acid. In the present study, we aimed to identify the unique characteristics of acetic acid responsible for this phenomenon. We found that one such feature is its hydrophobicity. We also discovered that acetic acid induces cell responses by intracellular acidification. Of the components of acetic acid in solution (protons, acetate ions, and undissociated acetic acid), undissociated acetic acid might be the functional unit that penetrates the lipid bilayer of cell membranes to acidify the intracellular environment, thereby inducing cell responses. The method used in this study might be convenient in evaluating the intracellular acidification of cultured cells by acids in the external environment.     

Regulation of Apoplastic pH in Source Leaves of Vicia faba by Gibberellic Acid

Leaf discs of broad bean (Vicia faba L.), peeled on the spongy mesophyll side, rapidly altered the pH of the surrounding medium (apoplast). Using pH indicator paper appressed against the leaf, immediately after peeling, initial apoplastic pH was estimated to be 4.5. Changes in the apoplastic pH were measured with a microelectrode placed into a 100-microliter drop of an unbuffered solution (2 millimolar KCl, 0.5 millimolar CaCl₂, and 200 millimolar mannitol) on the peeled surface. Discs acidified the medium until the pH stabilized at about 5.0 (about 10 minutes). Acidification was inhibited by 50 micromolar sodium vanadate, an inhibitor of the plasmalemma H⁺-ATPase and attenuated by omitting the osmoticum or potassium ions from the medium. Fusicoccin (10 micromolar) greatly enhanced the rate of acidification. The presence of 0.1 to 1 micromolar gibberellic acid resulted in a slower rate of medium acidification. Gibberellic acid appeared to modulate the activity of the H⁺-translocating ATPase located at the plasma membrane of the mesophyll cells.     

A comparison of the effects of 1,4-naphthoquinone and 2-hydroxy-1,4-naphthoquinone (lawsone) on indole-3-acetic acid (IAA)-induced growth of maize coleoptile cells

The effects of 1,4-naphthoquinone (NQ) and 2-hydroxy-1,4-naphthoquinone (NQ-2-OH) on indole-3-acetic acid (IAA)-induced growth, medium pH changes and membrane potential (E_m) in maize (Zea mays L.) coleoptile cells were determined. In addition, the redox cycling properties of both naphthoquinones were also compared. The dose-response curves constructed for the effects of NQ and NQ-2-OH on endogenous and IAA-induced growth differ in shape. It was found that NQ was by 10–50% more effective in inhibiting IAA-induced growth in maize coleoptile segments than NQ-2-OH. Simultaneous measurements of growth and external medium pH indicated that NQ and NQ-2-OH reduced or eliminated proton extrusion at all of the concentrations used, excluding NQ at 1 µM. It was found that both naphthoquinones at concentrations higher than 10 µM caused the depolarisation of the membrane potential (E_m). Additionally, compared to the controls, NQ- and NQ-2-OH-exposure of coleoptile segments, at concentrations higher than 10 µM, caused an elevation of the hydrogen peroxide (H₂O₂) production and plasma membrane redox activity. The highest catalase activity was observed at 10 µM NQ and it was ca. 18-fold greater (at 4 h) than in the control medium. Moreover, it was also found that NQ and NQ-2-OH, at all concentrations studied, increased the malondialdehyde content of coleoptile segments at 4 h of the experiment. The data presented here are discussed taking into account the “acid growth hypothesis” of auxin action and the mechanisms by which naphthoquinones interact with biological systems.     

THE EFFECT OF ACETATE BUFFER MIXTURES,ACETIC ACID,AND SODIUM ACETATE,ON THE PROTOPLASM,AS INFLUENCING THE RATE OF PENETRATION OF CRESYL BLUE INTO THE VACUOLE OF NITELLA

When living cells of Nitella are exposed to a solution of sodium acetate and are then placed in a solution of brilliant cresyl blue made up with a borate buffer mixture at pH 7.85, a decrease in the rate of penetration of dye is found, without any change in the pH value of the sap. It is assumed that this inhibiting effect is caused by the action of sodium on the protoplasm. This effect is not manifest if the dye solution is made up with phosphate buffer mixture at pH 7.85. It is assumed that this is due to the presence of a greater concentration of base cations in the phosphate buffer mixture. In the case of cells previously exposed to solutions of acetic acid the rate of penetration of dye decreases with the lowering of the pH value of the sap. This inhibiting effect is assumed to be due chiefly to the action of acetic acid on the protoplasm, provided the pH value of the external acetic acid is not so low as to involve an inhibiting effect on the protoplasm by hydrogen ions as well. It is assumed that the acetic acid either has a specific effect on the protoplasm or enters as undissociated molecules and by subsequent dissociation lowers the pH value of the protoplasm. With acetate buffer mixture the inhibiting effect is due to the action of sodium and acetic acid on the protoplasm. The inhibiting effect of acetic acid and acetate buffer mixture is manifested whether the dye solution is made up with borate or phosphate buffer mixture at pH 7.85. It is assumed that acetic acid in the vacuole serves as a reservoir so that during the experiment the inhibiting effect still persists.