Maize Coleoptile Cells Research Articles

Fusicoccin (FC)-Induced Rapid Growth, Proton Extrusion and Membrane Potential Changes in Maize (Zea mays L.) Coleoptile Cells: Comparison to Auxin Responses

The fungal toxin fusicoccin (FC) induces rapid cell elongation, proton extrusion and plasma membrane hyperpolarization in maize coleoptile cells. Here, these three parameters were simultaneously measured using non-abraded and non-peeled segments with the incubation medium having access to their lumen. The dose–response curve for the FC-induced growth was sigmoidal shaped with the maximum at 10−6 M over 10 h. The amplitudes of the rapid growth and proton extrusion were significantly higher for FC than those for indole-3-acetic acid (IAA). The differences between the membrane potential changes that were observed in the presence of FC and IAA relate to the permanent membrane hyperpolarization for FC and transient hyperpolarization for IAA. It was also found that the lag times of the rapid growth, proton extrusion and membrane hyperpolarization were shorter for FC compared to IAA. At 30 °C, the biphasic kinetics of the IAA-induced growth rate could be changed into a monophasic (parabolic) one, which is characteristic for FC-induced rapid growth. It has been suggested that the rates of the initial phase of the FC- and IAA-induced growth involve two common mechanisms that consist of the proton pumps and potassium channels whose contribution to the action of both effectors on the rapid growth is different.

Effects of Naphthazarin (DHNQ) Combined with Lawsone (NQ-2-OH) or 1,4-Naphthoquinone (NQ) on the Auxin-Induced Growth of Zea mays L. Coleoptile Segments.

Naphthoquinones, plants secondary metabolites are known for their antibacterial, antifungal, anti-inflammatory, anti-cancer and anti-parasitic properties. The biological activity of naphthoquinones is connected with their ability to generate reactive oxygen species and to modify biological molecules at their nucleophilic sites. In our research, the effect of naphthazarin (DHNQ) combined with 2-hydroxy-1,4-naphthoquinone (NQ-2-OH) or 1,4-naphthoquinone (1,4-NQ) on the elongation growth, pH changes of the incubation medium, oxidative stress and redox activity of maize coleoptile cells were investigated. This paper describes experiments performed with maize (Zea mays L.) coleoptile segments, which is a classical model system to study plant cell elongation growth. The data presented clearly demonstrate that lawsone and 1,4-naphthoquinone combined with naphthazarin, at low concentrations (1 and 10 nM), reduced the endogenous and IAA-induced (Indole-3-Acetic Acid) elongation growth of maize coleoptile segments. Those changes in growth correlated with the proton concentration in the incubation medium, which suggests that the changes in the growth of maize coleoptile segments observed in the presence of naphthoquinones are mediated through the activity of PM H+-ATPase. The presence of naphthoquinones induced oxidative stress in the maize coleoptile tissue by producing hydrogen peroxide and causing changes in the redox activity. Moreover, the incubation of maize segments with both naphthoquinones combined with naphthazarin resulted in lipid peroxidation and membrane damage. The regulation of PM H+-ATPase activity, especially its inhibition, may result from two major types of reaction: first, a direct interaction between an enzyme and naphthoquinone, which leads to the covalent modification of the protein thiols and the generation of thioethers, which have been found to alter the activity of the PM H+-ATPases; second, naphthoquinones induce reactive oxygen species (ROS) production, which inhibits PM H+-ATPases by increasing cytosolic Ca2+. This harmful effect was stronger when naphthazarin and 1,4-naphthoquinone were added together. Taking these results into account, it can be suggested that by combining naphthoquinones in small quantities, an alternative to synthetic pesticides could be developed.

The Effect of Naphthazarin on the Growth, Electrogenicity, Oxidative Stress, and Microtubule Array in Z. mays Coleoptile Cells Treated With IAA.

Naphthazarin (5,8-dihydroxy-1,4-naphthoquinone, DHNQ) is a naturally occurring 1,4-naphthoquinone derivative. In this study, we focused on elucidating the toxic effect of this secondary metabolite on the growth of plant cells. The dose–response curves that were obtained for the effects of DHNQ on endogenous and IAA-induced growth in maize coleoptile segments differ in shape; in the first case, it is linear, while in the presence of auxin it is bell-shaped with the maximum at 1 μM. It was found that DHNQ at almost all concentrations studied, when added to the incubation medium inhibited endogenous growth (excluding naphthazarin at 0.001 μM) as well as growth in the presence of IAA. Simultaneous measurements of the growth and external medium pH of coleoptile segments indicated that DHNQ diminished or eliminated proton extrusion at all of the concentrations that were used. Interestingly, the oxidative stress in maize coleoptile cells, which was measured as hydrogen peroxide (H2O2) production, catalase activity, redox activity and malondialdehyde (MDA) content, increased at the lower concentrations of DHNQ (<1 μM), thus suggesting a specific character of its action. It was also found that naphthazarin at concentration higher than 0.1 μM caused the depolarization of the membrane potential (Em). An analysis of the organization and anisotropy of the cortical microtubules showed that naphthazarin at all of the concentrations that were studied changed the IAA-induced transverse microtubule reorientation to an oblique reorientation. Our results indicate that naphthazarin diminished the growth of maize coleoptile cells by a broad spectrum of its toxic effects, thereby suggesting that naphthazarin might be a hypothetical component of new bioherbicides and biopesticides.

Single and combined effects of Cd and Pb on the growth, medium pH, membrane potential and metal contents in maize (Zea mays L.) coleoptile segments

The mechanisms of the toxic effects of Cd and Pb on plant cell growth are still poorly understood. In particular, little is known about their interactive effects, which usually occur in the environment. Moreover, the data that do exist in the literature are controversial. This study describes experiments that were performed with maize (Zea mays) coleoptile segments, which is a classical model system for studies of plant cell elongation growth. Cadmium and lead, which were added at 0.1 mM, reduced the endogenous and IAA-induced elongation growth of maize coleoptile cells. When both metals were added together or in sequence, their effect on IAA-induced growth was more toxic. The medium pH changes, which were measured simultaneously with growth, indicated that while Pb stopped IAA-induced proton extrusion, Cd only partially diminished it. Although Cd was generally more accumulated than Pb in the maize coleoptile segments, when IAA was added together with Pb, it significantly increased the accumulation of the metal. The short-term electrophysiological experiments showed that the addition of Cd caused the depolarisation of the membrane potential (Em), whereas Pb caused membrane hyperpolarisation. In the long-term electrophysiological experiments, it was found that the Cd-induced Em changes are complex. In conclusion, these results suggest that the effects of Cd and Pb as well as their combination on the elongation growth of maize coleoptile cells and the accumulation of the metals result, among others, from different ionic mechanisms by which each metal change the membrane potential of the 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 (Em) 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 (Em). 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.

Effects of juglone and lawsone on oxidative stress in maize coleoptile cells treated with IAA.

Naphthoquinones are secondary metabolites widely distributed in nature and produced by bacteria, fungi and higher plants. Their biological activity may result from induction of oxidative stress, caused by redox cycling or direct interaction with cellular macromolecules, in which quinones act as electrophiles. The redox homeostasis is known as one of factors involved in auxin-mediated plant growth regulation. To date, however, little is known about the crosstalk between reactive oxygen species (ROS) produced by quinones and the plant growth hormone auxin (IAA). In this study, redox cycling properties of two naphthoquinones, juglone (5-hydroxy-1,4-naphthoquinone) and lawsone (2-hydroxy-1,4-naphthoquinone), were compared in experiments performed on maize coleoptile segments incubated with or without the addition of IAA. It was found that lawsone was much more effective than juglone in increasing both H2O2 production and the activity of antioxidative enzymes (SOD, POX and CAT) in coleoptile cells, regardless of the presence of IAA. An increase in the activity of Cu/Zn-SOD isoenzymes induced by both naphthoquinones suggests that juglone- and lawsone-generated H2O2 was primarily produced in the cytosolic and cell wall spaces. The cell potential to neutralize hydrogen peroxide, determined by POX and CAT activity, pointed to activity of catalase as the main enzymatic mechanism responsible for degradation of H2O2 Therefore, we assumed that generation of H2O2, induced more efficiently by LW than JG, was the major factor accounting for differences in the toxicity of naphthoquinones in maize coleoptiles. The role of auxin in the process appeared negligible. Moreover, the results suggested that oxidative stress imposed by JG and LW was one of mechanisms of allelopathic action of the studied quinones in plants.

Auxin Binding Protein 1: A Red Herring After All?

The natural auxin indole-3-acetic acid is the first hormone identified in plants, and since it plays such a central role in plant growth and development, auxin has been the subject of intensive studies. A central question has been how the auxin signal is perceived by plant cells. The earliest experiments showed the presence of auxin binding particles at the plasma membrane (PM) and in the endoplasmic reticulum (ER) (Hertel et al., 1972). Screens for PM-localized auxin binding activities have led to the photo-affinity labeling and purification of Auxin Binding Protein 1 (ABP1) from maize coleoptile cells (Lobler and Klambt, 1985).

Effect of K+ and Ca2+ on the indole-3-acetic acid- and fusicoccin-induced growth and membrane potential in maize coleoptile cells.

The role of potassium (K(+)) and calcium (Ca(2+)) in the regulation of plant growth and development is complex and needs a diverse range of physiological studies. Both elements are essential for satisfactory crop production. Here, the effects of K(+) and Ca(2+) ions on endogenous growth and growth in the presence of either indole-3-acetic acid (IAA) or fusicoccin (FC) were studied in maize (Zea mays) coleoptiles. Membrane potentials of coleoptile parenchymal cells, incubated in media containing IAA, FC and different concentrations of K(+) and Ca(2+), were also determined. Growth experiments have shown that in the absence of K(+) in the incubation medium, both endogenous and IAA- or FC-induced growth were significantly inhibited by 0.1 and 1 mM Ca(2+), respectively, while in the presence of 1 mM K(+) they were inhibited only by 1 mM Ca(2+). At 10 mM K(+), endogenous growth and growth induced by either IAA or FC did not depend on Ca(2+) concentration. TEA-Cl, a potassium channel blocker, added 1 h before IAA or FC, caused a reduction of growth by 59 or 45 %, respectively. In contrast to TEA-Cl, verapamil, the Ca(2+) channel blocker, did not affect IAA- and FC-induced growth. It was also found that in parenchymal cells of maize coleoptile segments, membrane potential (Em) was strongly affected by the medium K(+), independently of Ca(2+). However, lack of Ca(2+) in the incubation medium significantly reduced the IAA- and FC-induced membrane potential hyperpolarization. TEA-Cl applied to the control medium in the same way as in growth experiments caused Em hyperpolarization synergistic with hyperpolarization produced by IAA or FC. Verapamil did not change either the Em of parenchymal cells incubated in the control medium or the IAA- and FC-induced membrane hyperpolarization. The data presented here have been discussed considering the role of K(+) uptake channels in regulation of plant cell growth.

Role of chloride ions in the promotion of auxin-induced growth of maize coleoptile segments.

The mechanism of auxin action on ion transport in growing cells has not been determined in detail. In particular, little is known about the role of chloride in the auxin-induced growth of coleoptile cells. Moreover, the data that do exist in the literature are controversial. This study describes experiments that were carried out with maize (Zea mays) coleoptile segments, this being a classical model system for studies of plant cell elongation growth. Growth kinetics or growth and pH changes were recorded in maize coleoptiles using two independent measuring systems. The growth rate of the segments was measured simultaneously with medium pH changes. Membrane potential changes in parenchymal cells of the segments were also determined for chosen variants. The question of whether anion transport is involved in auxin-induced growth of maize coleoptile segments was primarily studied using anion channel blockers [anthracene-9-carboxylic acid (A-9-C) and 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS)]. In addition, experiments in which KCl was replaced by KNO3 were also performed. Both anion channel blockers, added at 0·1 mm, diminished indole-3-acetic acid (IAA)-induced elongation growth by ~30 %. Medium pH changes measured simultaneously with growth indicated that while DIDS stopped IAA-induced proton extrusion, A-9-C diminished it by only 50 %. Addition of A-9-C to medium containing 1 mm KCl did not affect the characteristic kinetics of IAA-induced membrane potential changes, while in the presence of 10 mm KCl the channel blocker stopped IAA-induced membrane hyperpolarization. Replacement of KCl with KNO3 significantly decreased IAA-induced growth and inhibited proton extrusion. In contrast to the KCl concentration, the concentration of KNO3 did not affect the growth-stimulatory effect of IAA. For comparison, the effects of the cation channel blocker tetraethylammonium chloride (TEA-Cl) on IAA-induced growth and proton extrusion were also determined. TEA-Cl, added 1 h before IAA, caused reduction of growth by 49·9 % and inhibition of proton extrusion. These results suggest that Cl(-) plays a role in the IAA-induced growth of maize coleoptile segments. A possible mechanism for Cl(-) uptake during IAA-induced growth is proposed in which uptake of K(+) and Cl(-) ions in concert with IAA-induced plasma membrane H(+)-ATPase activity changes the membrane potential to a value needed for turgor adjustment during the growth of maize coleoptile cells.

Alteration of plasmalemma H+-ATPase activity in maize coleoptile cells at different age of seedlings

An irregular alteration of the activity and character of the distribution of plasmalemma H + -ATPase has been shown in parenchymal cells of the coleoptile subapical zone within the period from the third to fifth day of ethiolated maize seedling development. The study was carried out by the cytochemical method. The highest enzyme activity was determined in the cells of four-day-old coleoptiles. The revealed change in H + -ATPase activity does not correspond to the dynamics of the growth intensity of the elongation of coleoptile cells within the studied period of seedling development.

A shift in sensitivity to auxin within development of maize seedlings

The auxin-induced changes in cytosolic concentrations of Ca(2+) and H(+) ions were investigated in protoplasts from maize coleoptile cells at 3rd, 4th and 5th day of development of etiolated seedlings. The shifts in [Ca(2+)](cyt) and [H(+)](cyt) were detected by use of fluorescence microscopy in single protoplasts loaded with the tetra[acetoxymethyl]esters of the fluorescent calcium binding Fura 2, or pH-sensitive carboxyfluorescein, BCECF, respectively. Both the auxin-induced shifts in the ion concentrations were specific to the physiologically active synthetic auxin, naphthalene-1-acetic acid (1-NAA), and not to the non-active naphthalene-2-acetic acid (2-NAA). Regardless of the age of the seedlings, the rise in [Ca(2+)](cyt) was prior to the acidification in all investigated cases. The maximal acidification coincided with the highest amplitude of [Ca(2+)](cyt) change, but not directly depended on the concentration of 1-NAA. Within aging of the seedlings the amplitude of auxin-induced [Ca(2+)](cyt) elevation decreased. The shift in auxin-induced acidification was almost equal at 3rd and 4th day, but largely dropped at 5th day of development. The acidification was related to changes in the plasma membrane H(+)-ATPase activity, detected as phosphate release. The decrement in amplitude of both the tested auxin-triggered reactions well coincided with the end of the physiological function of the coleoptile. Hence the primary auxin-induced increase in [Ca(2+)](cyt), which is supposed to be an important element of hormone signal perception and transduction, can be used as a test for elucidation of plant cell sensitivity to auxin.

Ca(2)+-stimulated exocytosis in maize coleoptile cells.

Changes in membrane capacitance (C(m)) after photolysis of the caged Ca(2)+ compound dimethoxynitrophenamine were studied in protoplasts from maize coleoptiles. Changes in C(m) values resulting from increased concentrations of free Ca(2)+ in the cytoplasm ([Ca(2)+](cyt)) were interpreted as representing changes in [Ca(2)+](cyt)-sensitive exocytosis and endocytosis. A continuous increase in [Ca(2)+](cyt) resulted in a sigmoidal increase in C(m) values with a half-maximal concentration at approximately 1 microM. The steep increase in C(m) values was followed by a variable slow phase in changing C(m) values. When [Ca(2)+](cyt) increased at a rate of 0.6 micromol L(-)(1) sec(-)(1), the initial steep increase in C(m) values lasted approximately 5 to 10 sec. During this time, protoplasts increased in surface area by approximately 2.5%. The biphasic dynamics of [Ca(2)+](cyt)-stimulated increases in C(m) values can be described by a kinetic model containing two pools of vesicles with two [Ca(2)+](cyt)-sensitive steps in the exocytotic pathway.

Ca 2+ -Stimulated Exocytosis in Maize Coleoptile Cells

Ca 2+ -Stimulated Exocytosis in Maize Coleoptile Cells

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

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 (VM). 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 μM 1-naphthylene acetic acid (NAA) to coleoptiles causes a transient depolarization followed by a slow hyperpolarization of VM. However, in the presence of PEC, VM only depolarized in NAA. After PEC-treated coleoptiles were washed free of the enzyme, NAA caused only small fluctuations of VM. A similarly small VM 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.

Auxin augments conductance of K + inward rectifier in maize coleoptile protoplasts

Potassium is taken up by maize (Zea mays L.) coleoptile cells via a typical plant inward rectifier (K ir ). Sufficient conductance of this channel is essential in order to maintain auxin-stimulated cell elongation. It was therefore investigated whether the activity of this channel is subject to direct or indirect control by this growth hormone. Patch-clamp measurements of whole coleoptile protoplasts revealed no appreciable effect of externally applied 10 μM or 100 μM α-naphthaleneacetic acid (NAA) on the activity of K ir over test periods of ≥ 18 or ≥ 8 min, respectively. When, however, K ir was recorded in the cell-attached configiuration and 10 μM NAA administered to the bath medium, the conductance of K ir increased significantly in 13 out of 18 protoplasts over the control. This rise occurred at a fixed protoplast voltage after a lag period of less than 10 min and exhibited no voltage dependency. The absence of response to NAA of protoplasts in the whole-cell configuration indicates that auxin perception and channel control is linked via a soluble cytoplasmic factor and that this mediator is washed out or modified upon perfusion of the cytoplasm with pipette solution. To search for this expected diffusible factor the K ir current was recorded before and after elevation of Ca2+ and H+ in the cytoplasm. In the whole-cell configuration the increase in Ca2+ from a nanomolar value to >1 μM by means of Ca2+-release from the caged precursor Na2-DM-nitrophen left K ir unaffected. The whole-cell K ir conductance was also not affected upon addition of 10 mM Na+-acetate to the bath medium, an operation used to lower the cytoplasmic pH. This excludes a primary role for the known auxin-evoked rise in cytoplasmic Ca2+ and H+ in K ir activity. We postulate that another, as yet unknown, mechanism mediates the auxin-evoked stimulation of the number of active K ir channels in the plasma membrane.

An auxin-binding protein is localized to the plasma membrane of maize coleoptile cells: identification by photoaffinity labeling and purification of a 23-kda polypeptide.

Plasma membrane vesicles were isolated from maize (Zea mays L.) coleoptile tissue by aqueous two-phase partitioning and assayed for homogeneity by the use of membrane-specific enzymatic assays. Using 5-azido-[7-3H]indole-3-acetic acid ([3H]N3IAA), we identified several IAA-binding proteins with molecular masses of 60 kDa (pm60), 58 kDa (pm58), and 23 kDa (pm23). Using Triton X-114, we were able to selectively extract pm23 from the plasma membrane. We show that auxins and functional analogues compete with [3H]N3IAA for binding to pm23. We found that PAB130, a polyclonal antibody raised against auxin-binding protein 1 (ABP-1), recognized ABP-1 as well as pm23. This suggests that pm23 shares common epitopes with ABP-1. In addition, we identified an auxin-binding protein with a molecular mass of 24 kDa (pm24), which was detected in microsomal but not in plasma membrane vesicle preparations. Like pm23 this protein was extracted from membrane vesicles with Triton X-114. We designed a purification scheme allowing simultaneous purification of pm23 and pm24. Homogeneous pm23 and pm24 were obtained from coleoptile extracts after 7000-fold purification.

Cytoplasmic streaming affects gravity-induced amyloplast sedimentation in maize coleoptiles

Living maize (Zea mays L.) coleoptile cells were observed using a horizontal microscope to determine the interaction between cytoplasmic streaming and gravity-induced amyloplast sedimentation. Sedimentation is heavily influenced by streaming which may (1) hasten or slow the velocity of amyloplast movement and (2) displace the plastid laterally or even upwards before or after sedimentation. Amyloplasts may move through transvacuolar strands or through the peripheral cytoplasm which may be divided into fine cytoplasmic strands of much smaller diameter than the plastids. The results indicate that streaming may contribute to the dynamics of graviperception by influencing amyloplast movement.

Short-term effects of plant hormones on membrane potential and membrane permeability of dwarf maize coleoptile cells (Zea mays L. d 1) in comparison with growth responses

The membrane potential difference of dwarf maize coleoptile cells is increased by both 10(-5)moll(-1) gibberellic acid (GA3) and indoleacetic acid (IAA) a few minutes after application. A final level is reached after 10-20 min. The membrane permeability ratio P Na:P K is altered by both hormones during the first 15 min after application, indicating a rapid effect on the membrane. Elongation growth of coleoptile segments, however, is only stimulated by IAA. The auxin-induced growth as well as the auxin effect on membrane permeability depends on the calcium ion concentration of the medium. It is concluded that IAA acts via a proton extrusion pump that is electrically balanced by a potassium ion uptake, driven by the electromotive force of the pump. The mode of action of GA3 on elongation growth is assumed to involve a process that depends on the physiologic state of the tissue and/or metabolic energy.

Polar auxin transport and auxin-induced elongation in the absence of cytoplasmic streaming

When cytoplasmie streaming in oat and maize coleoptile cells is completely inhibited by cytochalasin B (CB), polar transport of auxin (indole-3-acetic acid) continues at a slightly reduced rate. Therefore, cytoplasmic streaming is not required for polar transport. Auxin induces elongation in CB-inhibited coleoptile and pea stem segments, but elongation rate is reduced about 40% by CB. Therefore, stimulation of cytoplasmic streaming cannot be the means by which auxin promotes cell elongation, but streaming may be beneficial to elongation growth although not essential to it. A more severe inhibition of elongation develops after several hours in CB. With coleoptiles this could be due to inhibition of sugar uptake; in pea tissue it may be due to permeability changes and cytoplasmic degeneration. CB does not disorganize or disorient microfilament bundles when it inhibits streaming in maize, but appears instead to cause hypercondensation of microfilament material.

Intracellular localization of the active process in polar transport of auxin

The cytoplasm of maize coleoptile cells was displaced to either the apical or basal ends of the cells by centrifuging (1750xg for 10 min) segments in which protoplasmic streaming had been stopped by pretreatment with cytochalasin B. Centrifugation toward the base of the segment promotes the subsequent basipetal transport of indole-3-acetic acid, whereas apical centrifugation dramatically inhibits this transport. Apical centrifugation neither promotes acropetal transport nor reverses the polarity of auxin transport. Experiments in which the amyloplasts were separated from the bulk of the cytoplasm indicate that the basipetal transport is independent of both the position and pressure exerted by the amyloplasts but is strongly dependent on the amount of cytoplasm at the basal end of the cells. These effects of centrifugation on auxin transport lead to the conclusion that the metabolic component of the transport is a polar secretion of auxin localized in the basal plasma membrane of each cell.