The effect of calcium antagonists and inhibitors of secretory processes on auxin-induced elongation and fine structure ofPisum sativum stem segments

Summary The treatment of dark grown pea stem segments with chelators of divalent cations (EGTA, EDTA, CTC), various Ca²⁺ antagonists (LaCl₃, A-23187, verapamil) and inhibitors of secretory processes (monensin, CB) reduced elongation in the presence of indole-3-acetic acid (IAA). Generally the inhibition increased with increasing concentrations of the substances. The timing of the responses can be correlated with maximum auxin-stimulated secretion of cell wall material. Examination of cell ultrastructure showed that changes in dictyosome activity could explain a reduced deposition of cell wall material and so cause inhibition of elongation.The inhibitors affected the morphology and vesiculation of the dictyosomes, and the appearance of the plasma membrane, ER and mitochondria in different ways. The most pronounced effects on ultrastructure resulted from monensin and LaCl₃ treatments with the dictyosomes being most affected; large vesicles appeared in the cytoplasm. Less pronounced effects on cell structure were seen in EGTA, A-23187 and verapamil treated tissue. The effects on the dictyosomes are considered to be due to disturbances of Ca²⁺ and other ionic levels within the cells.Abbreviations ATPase adenosine triphosphatase - CB cytochalasin B - CTC chlorotetracycline - DMSO dimethyl sulphoxide - EDTA ethylene diamine tetraacetic acid - EGTA ethyleneglycol-bis-(B-amino ethyl ether)N,N¹-tetraacetic acid - ER endoplasmic reticulum - ver verapamil     

Relationship between IAA-induced elongation growth and plasma membrane NADH oxidase in soybean (Glycine max Merr.) hypocotyls

A relationship between the activity of NADH oxidase of the plasma membrane and the IAA-induced elongation growth of hypocotyl segments in etiolated soybean (Glycine max Merr.) seedlings was investigated. The plasma membrane NADH oxidase activity increased in parallel to IAA effect on elongation growth in hypocotyl segments. Actually, NADH oxidase activity was stimulated 3-fold by 1 u,M IAA, and the elongation rate of segments was stimulated 10-fold by 10 iM IAA. The short-term elongation growth kinetics, however, showed that the IAA-induced elongation of hypocotyl segments was completely inhibited by plasma membrane redox inhibitors such as actinomycin D and adriamycin, at 80 μM and 50 μM respectively. In addition, 1 mM actinomycin D inhibited the IAA-stimulated NADH oxidase activity by about 80%. However, adriamycin had no effect on NADH oxidase activity of plasma membrane vesicles. Based on these results, the plasma membrane redox reactions seemed to be involved in IAA-induced elongation growth of hypocotyls, and the redox component responding to IAA was suggested to be NADH oxidase.     

The effect of cytochalasin B on the rate of growth and ultrastructure of wheat coleoptiles and maize roots

Cytochalasin B (CB) inhibits the elongation growth of maize roots, and that of wheat coleoptile segments incubated in indolyl-3-acetic acid, by over 30% after a lag period of about 60 min. This long lag is not due to poor tissue penetration by the inhibitor, but seems to reflect a property of the process inhibited by CB. The only visible ultrastructural change accompanying growth inhibition is the accumulation of secretory vesicles in the vicinity of dictyosomes, which occurs between 90 and 300 min. However, a massive accumulation of vesicles is seen after 120 min in root cap cells which possess very active dictyosomes. The results indicate that CB does not inhibit elongation growth by interfering with cytoplasmic streaming. Instead, they indicate that the drug acts to inhibit the secretion of cell wall components at some stage after vesicle production, but prior to their transport.Abbreviations CB cytochalasin B - IAA indolyl-3-acetic acid - DMSO dimethyl sulphoxide     

Occurrence of osmiophilic particles is correlated to elongation growth of higher plants

Summary The occurrence of elongation growth-related osmiophilic particles (OPs) was investigated in hypocotyls of sunflower, bean, and spruce as well as in pea epicotyls and in cress roots of intact seedlings. In all analyzed species, OPs were found to occur specifically within the periplasmic space between plasma membrane and the outer epidermal cell walls of elongating parts of hypocotyls, epicotyls, and roots, whereas cells of nonelongating parts were devoid of OPs. Auxin (IAA) markedly increased the number of OPs in epicotyl and hypocotyl segments. Treatment of pea epicotyl segments with the lectin concanavalin A inhibited their elongation growth in the presence of IAA. At a subcellular level this effect was characterized by the occurrence of a pronounced osmiophilic layer in the periplasmic space of the outer periclinal and the outer part of the anticlinal epidermal cell walls. Treatment of IAA-incubated segments with the secretion inhibitor brefeldin A inhibited both elongation growth and periplasmic occurrence of OPs. This effect was accompanied by complementary accumulation of OPs in the peripheral cytoplasm of epidermal cells. Together the results indicate that IAA-induced epidermis-specific secretion of OPs is closely related to cell elongation growth not only in organs of monocotyledonous species, but also in dicotyledonous angiosperms as well as in gymnosperms.Abbreviations OPs osmiophilic particles - ConA concanavalin A - BFA brefeldin A - IAA -indolyl acetic acid     

Transport of cholesterol from the endoplasmic reticulum to the plasma membrane

We have studied the transport of newly synthesized cholesterol from the endoplasmic reticulum to the plasma membrane in Chinese hamster ovary cells using a cell fractionation assay. We found that transport is dependent on metabolic energy, but that the maintenance of the high differential concentration of cholesterol in the plasma membrane is not an energy-requiring process. We have tested a variety of inhibitors for their effect on cholesterol transport and found that cytochalasin B, colchicine, monensin, cycloheximide, and NH4Cl did not have any effect. The cholesterol transport process shows a sharp temperature dependence; it ceases at 15 degrees C, whereas cholesterol synthesis continues. When synthesis occurs at 15 degrees C, the newly synthesized cholesterol accumulates in the endoplasmic reticulum and in a low density, lipid-rich vesicle fraction. These results suggest that cholesterol is transported via a vesicular system.     

Flower stalk segments of Arabidopsis thaliana ecotype Columbia lack the capacity to grow in response to exogenously applied auxin

Exogenously applied IAA stimulated cell elongation of segments excised from flower stalks of Arabidopsis thaliana ecotype Landsberg erecta (Ler) by increasing the cell wall extensibility, but it did not affect that of ecotype Columbia (Col). Treatment with a low pH buffer solution (pH 4.0) or fusicoccin (FC), a reagent activating H(+)-ATPases, significantly increased the cell wall extensibility and promoted elongation growth of flower stalk segments of both ecotypes, indicating that the flower stalk segments of Col possess the capacity to grow under acidic pH conditions. IAA promoted the proton excretion in segments of Ler but not of Col. On the other hand, FC increased the proton excretion in segments of Col as much as that of Ler. These results suggest that IAA activates the plasma membrane H(+)-ATPases in the segments of Ler but not those of Col, while FC activates them in both ecotypes. Flower stalks of Col may lack the mechanisms of activation by IAA of the plasma membrane H(+)-ATPases.     

Plasma membrane phospholipid and sterol synthesis in soybean hypocotyl segments undergoing auxin-induced elongation

Summary Auxin-induced cell elongation necessitates plasma membrane enlargement. The effect of auxin (10 M 2,4-dichlorophenoxyacetic acid) treatment on amount, composition, and rate of synthesis of plasma membrane lipids was examined. Auxin-treated and control soybean (Glycine max L.) hypocotyl segments were incubated with [¹⁴C]acetate for times ranging from 0.5 to 18 h, prior to isolation of plasma membrane by aqueous two-phase partitioning. The composition of individual plasma membrane lipids in elongating segments did not differ from the composition in treatment time-matched control segments, except that after longer auxin treatments, phospholipids had more unsaturated fatty acids. Plasma membrane phospholipid and free sterol content both increased in elongating segments. The relative proportion of sterols and phospholipids in the plasma membrane primarily depended on time after segment excision, for both auxin-treated and control segments. Auxin enhanced the rate of lipid incorporation into the plasma membrane by 6 h, and stimulated the synthesis of some phospholipids and sterols.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - ER endoplasmic reticulum - GC gas chromatography - IAA indole-3-acetic acid - PA phosphatidic acid - PC phosphatidylcholine - PE phosphatidylethanolamine - PG phosphatidylglycerol - PI phosphatidylinositol - PM plasma membrane - PS phosphatidylserine     

Requirement of RNA for the Auxin-induced Elongation of Oat Coleoptile

Using etiolated oat coleoptile segments the following results were obtained. Actinomycin D pretreatment for one hour produced about 50 per cent inhibition of RNA synthesis (labeled uracil incorporation), but the elongation caused by IAA was not inhibited in the following 5 hours at least. Actinomycin D pretreatment for three hours produced about 75 per cent inhibition of RNA synthesis and almost complete inhibition of subsequent IAA-induced elongation, which is accompanied by the inhibition of IAA-induced increase in cell wall extensibility. The inhibiting effect of actinomycin D seemed to be reduced when IAA was given within a certain period.     

Estimation of Golgi membrane flow rates in ovary glands of aptenia cordifolia using cytochalasin B

Ovary gland cells of Aptenia cordifolia were exposed to 100 micrograms/ml cytochalasin B (cyt B) for 30 or 60 min during the phase of granulocrine polysaccharide secretion. The drug caused a congestion of Golgi vesicles around the dictyosomes, probably resulting from an inhibition of the vesicle migration towards the plasma membrane. The ultrastructural feature of the Golgi apparatus in control and cyt B treated cells was analyzed using stereological methods in order to estimate the mean area of vesicular membrane produced by a single dictyosome during a 30 min period of effective cyt B action. Assuming that the rate of vesicle congestion can be equated with the rate of vesicle production, the 236 dictyosomes found to be present in the non-growing ovary gland cells form 7517 vesicles in total, or approximately 32 vesicles each within a period of 30 min. This corresponds to a membrane turnover rate of 70.4 micrometers/min (this equals approximately 10% of the total plasma membrane area per min), since the mean vesicle surface area was calculated to be 0.281 microns2. The turnover time of a single Golgi cisterna was determinated to be 7.34 min, and the average vesicle life time to be 8.86 min. Discussion focuses upon the way by which the relatively high amount of vesicular membrane material incorporated into the plasmalemma is recycled into the endomembrane system. Since a bulk membrane retrieval in the form of vesicles, as well as a bulk vesicle migration from the ER to the dictyosomes could not be observed, we suggest that a transfer of membrane subunits is involved in the maintenance of membrane equilibrium in the Golgi apparatus.     

Time-dependent Changes in the Auxin Sensitivity of Coleoptile Segments: Apparent Sensory Adaptation

When segments are excised from corn (Zea mays L.) coleoptiles they exhibit a very low rate of elongation for about 3.5 hours. A strong increase in growth rate (the spontaneous growth response) then occurs and persists for many hours. During the latent period preceding the spontaneous growth response there is an apparent increase with time in the sensitivity of the segments to indoleacetic acid (IAA). This increase in sensitivity is expressed as a 2- to 3-fold increase in the magnitude of the growth response to low levels of IAA and a 3-fold decrease in the latent period of the response during the first 3 hours following excision. A similar increase in sensitivity to low levels of IAA is noted if application of IAA is timed from the point of termination of a previous exposure to the hormone. Since the increase in responsiveness to low levels of IAA is not paralleled by an increase in the rate of uptake of the hormone, the data may be interpreted as evidence for a type of time-dependent sensory adaptation to auxin. The IAA dose-response relationship also changes with time, and there is indirect evidence that an auxin-dependent inhibitor may influence the expression of the apparent sensory adaptation to auxin.     

Vesicle swelling regulates content expulsion during secretion

The involvement of secretory vesicle swelling has been proposed in secretion; however, little is known about its role. Using both the pancreatic acinar cell and neuronal model, we show secretory vesicle swelling in live cells. Our study reveals that vesicle swelling potentiates its fusion at the cell plasma membrane, and is required for expulsion of intravesicular contents. Since the extent of swelling is directly proportional to the amount of vesicular contents expelled, this provides cells with the ability to regulate release of secretory products. These direct observations of the requirement of secretory vesicle swelling in secretion, provides an understanding of the appearance of partially empty vesicles following the process.     

Membrane trafficking and osmotically induced volume changes in guard cells

Guard cells rapidly adjust their plasma membrane surface area while responding to osmotically induced volume changes. Previous studies have shown that this process is associated with membrane internalization and remobilization. To investigate how guard cells maintain membrane integrity during rapid volume changes, the effects of two membrane trafficking inhibitors on the response of intact guard cells of Vicia faba to osmotic treatments were studied. Using confocal microscopy and epidermal peels, the relationship between the area of a medial paradermal guard-cell section and guard-cell volume was determined. This allowed estimates of guard-cell volume to be made from single paradermal confocal images, and therefore allowed rapid determination of volume as cells responded to osmotic treatments. Volume changes in control cells showed exponential kinetics, and it was possible to calculate an apparent value for guard-cell hydraulic conductivity from these kinetics. Wortmannin and cytochalasin D inhibited the rate of volume loss following a 0-1.5 MPa osmotic treatment. Cytochalasin D also inhibited volume increases following a change from 1.5 MPa to 0 MPa, but wortmannin had no effect. Previous studies showing that treatment with arabinanase inhibits changes in guard-cell volume in response to osmotic treatments were confirmed. However, pressure volume curves show that the effects of arabinanase and the cytochalasin D were not due to changes in cell wall elasticity. It is suggested that arabinanase, cytochalasin D, and wortmannin cause reductions in the hydraulic conductivity of the plasma membrane, possibly via gating of aquaporins. A possible role for aquaporins in co-ordinating volume changes with membrane trafficking is discussed.     

Actin is required for endocytic trafficking in the malaria parasite Plasmodium falciparum

The intra-erythrocytic stages of the malaria parasite endocytose large quantities of the surrounding erythrocyte cytoplasm and deliver it to a digestive food vacuole via endocytic vesicles. Digestion provides amino acids for parasite protein synthesis and is required to maintain the osmotic integrity of the host cell. The parasite endocytic pathway has been described morphologically by electron microscopy, but the molecular mechanisms that mediate and regulate it remain elusive. Given the involvement of actin in endocytosis in other eukaryotes, we have used actin inhibitors to assess the requirement for this protein in the endocytic pathway of the human malaria parasite, Plasmodium falciparum . Treatment of cultures with cytochalasin D did not affect haemoglobin levels in the parasites when co-administered with protease inhibitors, and neither did it affect the uptake of the endocytic tracer horseradish peroxidase, suggesting the absence of actin in the mechanism of endocytosis. However, in the absence of protease inhibitors, treated parasites contained increased levels of haemoglobin due to an accumulation of enlarged endocytic vesicles, as determined by immunofluorescence and electron microscopy, suggesting a role for actin in vesicle trafficking, possibly by mediating vesicle maturation and/or fusion to the digestive vacuole. In contrast to cytochalasin D, treatment with jasplakinolide led to an inhibition of endocytosis, an accumulation of vesicles closer to the plasma membrane and a marked concentration of actin in the parasite cortex. We propose that the stabilization of cortical actin filaments by jasplakinolide interferes with normal endocytic vesicle formation and migration from the cell periphery.     

Thymic hormone-containing cells. VIII. Effects of colchicine, cytochalasin B, and monensin on secretion of thymulin by cultured human thymic epithelial cells

The intracellular pathway for secretion of thymulin, a thymic hormone, was studied in primary cultures of human thymic epithelial cells by experimentally blocking the movement of secretory vesicles within these cells. These cultures were subjected to cytoskeleton inhibitors, such as colchicine and/or cytochalasin B, that block the directed movement of secretory vesicles, or to monensin, an ionophore that specifically perturbs the traffic of Golgi-derived vesicles. Both cytoskeleton inhibitors partially prevented thymulin secretion into the culture supernatants, and their effects were dose-dependent. Moreover, the percentage of thymulin-containing cells (evaluated by immunofluorescence with a zinc-specific anti-thymulin monoclonal antibody), as well as the fluorescence intensity within these cells, was significantly higher than observed in control cultures, suggesting that the hormone was accumulated in the cytoplasm, thus facilitating its detection. Similar results were obtained with monensin. These results, together with the recent identification of high molecular weight proteins reacting with anti-thymulin antibodies, suggest that thymulin is secreted via the following intracellular pathway: a precursor is synthesized at the level of the granular endoplasmic reticulum; it migrates to the Golgi complex, from which it is released within hormone-containing vesicles; the vesicles incorporate zinc, move towards the cell membrane, and eventually fuse with it. This sequence of events characterizes the classical phenomenon of exocytosis.     

Plant cell extension: structural implications for the origin of the plasma membrane

Abstract. The proposal that rapidly elongating plant cells cannot maintain plasma membrane synthesis by means of the normal endomembrane system has been examined in elongating segments of Avena sativa coleoptiles. Segments were sampled and fixed for electron microscopy, before and after elongation on auxin solutions. Mean cell extensions, cytoplasmic volumes, dictyosomc numbers, and vesicle sizes and numbers were determined. It was shown that there are sufficient dictyosomes present to sustain the vesicle production necessary for the observed plasma membrane extension.     

The roles of cell-wall acidification and proton-pump stimulation in auxin-induced growth: studies using monensin

The carboxylic ionophore, monensin, rapidly induced cell-wall acidification and a decrease in cytosolic pH when added to maize coleoptiles at low external pH and Na⁺ concentration. Elongation growth at rates equivalent to those obtained with indole-3-acetic acid was induced for about 1 h. Stimulation of the outwardly directed proton pump apparently occurred, since under the same conditions monensin induced membrane hyperpolarization of maize root rhizodermis cells. When the external pH was high (>8) and Na⁺ present, monensin treatment caused only minimal changes in membrane potential and cytosolic pH. Although the ionophore transported protons out of the cell, resulting in cell-wall acidification, no elongation growth occurred. However, under identical conditions, indole-3-acetic acid dit induce growth. The data indicates that stimulation of the outwardly directed electrogenic proton pump rather than the subsequent acidification of the cell wall is vital for the induction of elongation growth.Abbreviations CFA₂ 6-carboxyfluorescein diacetate - FA₂ fluorescein diacetate - Hepes 4-(2-hydroxyethyl-1-piperazinepropanesulfonic acid - IAA indole-3-acetic acid - Mes 2-(N-morpholino) ethanesulfonic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Stress relaxation properties of the cell wall of tissue segments under different growth conditions

Stress-relaxation parameters were compared under different experimentalconditions using 5th internode segments of light-grown pea seedlingsand coleoptile segments of dark-grown Avena seedlings. The followingresults were obtained. 1. In a short incubation period at 25?C, IAA caused a decreasein the minimum relaxation time, To, of the epidermal cell wallof pea internodes when it induced elongation; the optimum concentrationof IAA for decreasing To was 10 mg/liter. 2. At all concentrations of IAA used, 0.1–1000 mg/liter,the relationship between the To value of the epidermal cellwall peeled from segments incubated for 2 hr and the subsequentelongation rate in 2–3 hr incubation was linear, indicatingthat the To value of the cell wall at a certain time regulatesthe rate of the following elongation. 3. When segments of pea epicotyls or Avena coleoptiles wereincubated in mannitol solution of various concentrations inthe presence and absence of IAA and then allowed to grow inthe absence of both mannitol and IAA, the segments extendeddifferently depending upon the mannitol concentration, whichwas less than 0.3 M, given during preincubation. 4. The T_o and b (relaxation rate, S/log t) values were smallerin the cell wall of segments which extended more, than in thosewhich extended less. In this case, 0.2 M mannitol solution wasmost effective, since it inhibited IAA-induced elongation duringpre-incubation and the segments thus incubated extended themost afterward. 5. Extensibility, mm/gr, seemed to parallel the elongation whichhad occurred during pre-incubation, indicating that this value,contrary to To, represented at least partly the result of elongation. From these results we concluded that the growth rate to followis regulated by the minimum stress relaxation time, To, andpossibly by the relaxation rate, b, of the cell wall beforeextension, and these parameters may represent certain biochemicalmodifications of the cell wall components needed for cell extension. (Received August 12, 1974; )     

表油菜素内酯促进小麦胚芽鞘伸长的作用

BR促进小麦胚芽鞘伸长的生理活性大于IAA,但高浓度的促进现象不如IAA明显。BR刺激乙烯生成与浓度相关。BR和IAA混合处理,对芽鞘切段的伸长、乙烯释放和H~+分泌都表现了加成作用。这二种激素在作用时间上有明显的差别,BR作用的滞后期更为清楚。BR有拮抗ABA对小麦胚芽鞘切段伸长的抑制作用。     

Plasma membrane protein composition and synthesis in soybean hypocotyl segments undergoing auxin-induced elongation

Summary The types and amount of plasma membrane proteins synthesized during cell elongation in response to auxin (2,4-dichlorophenoxyacetic acid) treatment were investigated. Auxin-treated and control soybean (Glycine max L.) hypocotyl segments were incubated with [³⁵S]methionine for various times, ranging from 0.5 to 18 h, prior to isolation of plasma membrane by aqueous two-phase partitioning. Protein accumulated in the plasma membrane after auxin treatment. Despite this accumulation, the protein incorporation rate, estimated by the amount of label in the plasma membrane following a 0.5 h [³⁵S]methionine pulse, was unaffected by auxin treatment at both 0.5 and 18 h of treatment. Protein apparently accumulated by a mechanism distinct from enhanced incorporation. The plasma membrane proteins synthesized by elongating segments differed from controls at 18 h, as evidenced by the pattern of fluorographs following a 0.5 h radiolabelling. However, auxin treatment did not alter the 2-D gel pattern of the polypeptides detectable by silver stain.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IEF isoelectric focusing - PM plasma membrane - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Role of protein and RNA synthesis in the initiation of auxin-mediated growth in coleoptiles of Zea mays L.

The kinetics of inhibition by protein- and RNA-synthesis inhibitors (cycloheximide and cordycepin, respectively) of indole-3-acetic acid (IAA)-induced elongation growth were investigated using abraded coleoptile segments of Zea mays L. Removal of the cuticle — a diffusion barrier for solutes — by mechanical abrasion of the outer epidermal cell wall increased the effectiveness of inhibitors tremendously. In an attempt to elucidate the role of growth-limiting protein(s) (GLP) in the growth mechanism the following results were obtained. The elongation induced by IAA was completely inhibited when cycloheximide (10 mol·l^-1) was applied to abraded coleoptile segments as shortly as 10 min before the onset of the growth response (=5 min after administration of IAA). However, when cycloheximide was applied after 60 min of IAA treatment (when a steady-state growth rate is reached), the time required for complete cessation of growth was much longer (about 40 min). Cycloheximide inhibited the incorporation of [³H]leucine into protein within about 5 min. Cordycepin (400 mol·l^-1) prevented IAA-induced growth when applied as shortly as 25 min before the onset of the growth response (=10 min before administration of IAA) but required more than 60 min for a full inhibition of steady-state growth. The incorporation of [³H]adenosine into RNA was inhibited by cordycepin within 10 min. It is concluded that, contrary to previous investigations with nonabraded organ segments, the initiation of growth by IAA depends directly on the synthesis of GLP. Moreover, the apparent lifetime of GLP is at least four times longer than the time required by cycloheximide to inhibit the initiation of growth by IAA. This is interpreted to mean that GLP is not present before IAA starts to act but is synthesized as a consequence of IAA action starting a few minutes before the initiation of growth. Interpreting the kinetics of growth inhibition by cordycepin in a similar way, we further conclude that GLP synthesis is mediated by IAA-induced synthesis of the corresponding mRNA which starts about 10 min before the onset of GLP synthesis. Inhibition by cycloheximide and cordycepin of IAA-induced growth cannot be alleviated by acidifying the cell wall to pH 4-5, indicating that these inhibitors do not act on growth via an inhibition of auxin-mediated proton excretion.Abbreviations CHI cycloheximide - COR cordycepin - GLP growth-dimiting protein(s) - IAA indole-3-acetic acid - mRNA_GLP mRNA coding for GLP