Fusicoccin structure-activity relationships: Stimulation of growth by cell enlargement and promotion of seed germination

The activity of a number of fusicoccin derivatives and analogues has been assayed on growth by cell enlargement and on proton extrusion in pea ( Pisum sativum L, cv. Alaska) internode segments, on growth by cell enlargement in isolated squash ( Cucurbita maxima Dechesne in Lam. variety Mantovana) cotyledons, and on germination of light-requiring lettuce ( Lactuca sativa L. cv. Grand Rapids) and of abscisic acid-inhibited radish ( Raphanus sativus L. variety Tondo Rosso Quarantino) seeds. A similar pattern of activity in the different tests was found for most fusicoccin derivatives and analogues. As with fusicoccin, the activity of its derivatives and analogues on growth of pea internodes is paralleled by the activity on the capability of the tissue to acidify the incubation medium.
The obtained data are in full agreement with the view that the activity of fusicoccin on different physiological processes depends on its primary activation of a single system, involved in proton extrusion.     

Effect of fusicoccin and gibberellic acid on primary dormant Avena fatua caryopses

Fusicoccin induced germination in dormant and partially afterripened dormant caryopses of Avena fatua L. The rate of caryopsis germination was slower and final percentage germination lower in the highly dormant inbred line M73 at a given concentration of fusicoccin than in the dormant caryopses of line AN265. Gibberellic acid was more effective than fusicoccin in breaking dormancy in both lines. Promotion of germination of dormant caryopses by fusicoccin was inhibited by a 6-day pretreatment with (2-chloroethyl)trimethylammonium chloride.
The basal rate of proton efflux from embryos isolated from dormant and fully afterripened line AN265 caryopses was similar. Addition of fusicoccin increased the rate of proton efflux from the isolated embryos of dormant and afterripened caryopses by nearly 400%. Gibberellic acid had no effect on the rate of proton extrusion. The uptake of ⁸⁶Rb⁺ in dormant and afterripened A. fatua embryos was similar after a 2 h uptake period. The addition of fusicoccin to the medium doubled the uptake of ⁸⁶Rb⁴ by dormant and afterripened embryos. Gibberelleic acid had no effect on the uptake of ⁸⁶Rb⁺ by isolated embryos from either dormant or afterripened caryopses. The experimental results indicate that gibberellic acid is more versatile in its action than fusicoccin, and gibberellic acid may facilitate dormant A. fatua caryopsis germination by stimulating mechanisms other than the direct H⁺ efflux and K⁺ uptake at the membrane level.     

Action of protein synthesis inhibitors in blocking electrogenic h efflux from corn roots

The block in the electrogenic H⁺ efflux produced by protein synthesis inhibitors in corn root tissue can be released or by-passed by addition of fusicoccin or nigericin. The inhibition also lowers cell potential, and the release repolarizes. Associated with the inhibition of H⁺ efflux is inhibition of K⁺ influx and the growth of the root tip; fusicoccin partially relieves these inhibitions, but nigericin does not. The inhibition of H⁺ efflux which arises from blocking the proton channel of the ATPase by oligomycin or N,N′-dicyclohexylcarbodiimide can also be partially relieved by fusicoccin, but not by nigericin; the inhibition produced by diethylstilbestrol is not relieved by fusicoccin.     

ACID-INDUCED FILAMENT ELONGATION IN IPOMOEA NIL (CONVOLVULACEAE)

It has been known for some time that morning glory filaments elongate in response to increases in concentration of gibberellins (Murakami, 1973) and decreases in ethylene production (Koning and Raab, in press), but many other aspects of their growth have remained unstudied. In the present work, the possible role of gibberellin-stimulated proton efflux in filament growth was examined. Although applied gibberellins stimulated extensive filament growth in vitro and the pH of the incubating medium became acidified during growth, gibberellin also induced growth in media buffered at alkaline pH values. Acidic buffers alone elicited only a very small amount of growth. Fusicoccin, a potent stimulator of proton efflux, initially stimulated the rate of filament growth but elicited only a small increment of growth. In fact, continued presence of fusicoccin poisoned sustained gibberellin-induced growth. Vanadate ions, believed to inhibit proton efflux, had little effect upon gibberellin-induced growth except at extremely high concentrations. Based upon these results, it appears that the acid-induced component of growth stimulation by gibberellin is relatively minor in Ipomoea filaments. These results are quite different from those reported for filament elongation in Gaillardia (Koning, 1983a).     

On the Nature and Origin of the Calcium Asymmetry Arising during Gravitropic Response in Etiolated Pea Epicotyls

Seven day old etiolated pea epicotyls were loaded symmetrically with ³H-indole 3-acetic acid (IAA) or ⁴⁵Ca²⁺, then subjected to 1.5 hours of 1g gravistimulation. Epidermal peels taken from top and bottom surfaces after 90 minutes showed an increase in IAA on the lower side and of Ca²⁺ on the upper side. Inhibitors of IAA movement (TIBA, 9-hydroxyfluorene carboxylic acid) block the development of both IAA and Ca²⁺ asymmetries, but substances known to interfere with normal Ca²⁺ transport (nitrendipine, nisoldipine, Bay K 8644, A 23187) do not significantly alter either IAA or Ca²⁺ asymmetries. These substances, however, are active in modifying both Ca²⁺ uptake and efflux through oat and pea leaf protoplast membranes. We conclude that the ⁴⁵Ca²⁺ fed to pea epicotyls occurs largely in the cell wall, and that auxin movement is primary and Ca²⁺ movement secondary in gravitropism. We hypothesize that apoplastic Ca²⁺ changes during graviresponse because it is displaced by H⁺ secreted through auxin-induced proton release. This proposed mechanism is supported by localized pH experiments, in which filter paper soaked in various buffers was applied to one side of a carborundum-abraded epicotyls. Buffer at pH 3 increases calcium loss from the side to which it is applied, whereas pH 7 buffer decreases it. Moreover, 10 micromolar IAA and 1 micromolar fusicoccin, which promote H⁺ efflux, increase Ca²⁺ release from pea epicotyl segments, whereas cycloheximide, which inhibits H⁺ efflux, has the reverse effect. We suggest that Ca²⁺ does not redistribute actively during gravitropism: the asymmetry arises because of its release from the wall adjacent to the region of high IAA concentration, proton secretion, and growth. Thus, the asymmetric distribution of Ca²⁺ appears to be a consequence of growth stimulation, not a critical step in the early phase of the graviresponse.     

Proton efflux from oat coleoptile cells and exchange with wall calcium after IAA or fusicoccin treatment

Elongation growth of plant cells occurs by stretching of cell walls under turgor pressure when intermolecular bonds in the walls are temporarily loosened. The acid-growth theory predicts that wall loosening is the result of wall acidification because treatments (including IAA and fusicoccin) that cause lowered wall pH cause elongation. However, conclusive evidence that IAA primarily reduces wall pH has been lacking. Calcium has been reported to stiffen the cell walls. We have used a microelectrode ion-flux measuring technique to observe directly, and non-invasively, the net fluxes of protons and calcium from split coleoptiles of oats (Avena sativa L.) in unbuffered solution. Normal net fluxes are 10 nmol · m⁻² · s⁻¹ proton efflux and zero calcium flux. The toxin fusicoccin (1 μM) causes immediate efflux from tissue not only of protons, but also of calcium, about 110 nmol · m⁻² · s⁻¹ in each case. The data fit the “weak acid Donnan Manning” model for ion exchange in the cell wall. Thus we associate the known “acid-growth” effect of fusicoccin with the displacement of calcium from the wall by exchange for protons extruded from the cytoplasm. Application of 10 μM IAA causes proton efflux to increase transiently by about 15 nmol · m⁻² · s⁻¹ with a lag of about 10 min. The calcium influx decreases immediately to an efflux of about 20 nmol · m⁻² · s⁻¹. It appears that auxin too causes an “acid-growth” effect, with extruded protons exchanging for calcium in the cell walls. I. Arif is currently recieving an AIDAB scholarship. This work was supported by an Australian Research Council grant to I.A. Newman.     

Proton Efflux from Roots of Intact Spergularia marina Plants

Despite numerous considerations of the importance of protonextrusion to plant mineral nutrition, measurements of net H⁺efflux rates in photo-autotrophic plants growing in completemedium are rare, and the rates of efflux reported are considerablylower than that typical of low-salt seedlings. We present herethe first report of short term net proton efflux from rootsof a halophyte. and the highest rate of net efflux reportedto date for any higher plant, in both low-salt and completenutrient media. We also show responses of that efflux to a varietyof treatments, including NaCl fusicoccin, shoot excision anddarkness. Key words: Halophyte, Sea spurrey, H⁺ efflux, Roots     

Some physiological effects of podolactone-type inhibitors

Podolactone-type inhibitors, including harringtonolide and lycoricidinol, affect several bioassays at 1 to 10 μ M. Although these compounds were less active than abscisic acid in inhibiting growth of coleoptiles of wheat embryos, podolactone A significantly decreased the number of mature spikelets of Lolium temulentum at a lower concentration than abscisic acid. Lycoricidinol was more active than podolactone E, the most active of the podolactones, in three bioassays. Inhibition of growth induced by gibberellic acid or zeatin with podolactone-type compounds was not competitive. Harringtonolide was a strong inhibitor of growth at higher concentrations but did not affect auxin transport, while podolactone E and lycoricidinol inhibited it. Lycoricidinol, podolactone E and harringtonolide did not retard senescence of leaf-discs. The promotive effects of gibberellic acid in the barley endosperm bioassay were counteracted by podolactone A.     

Evidence that H+ efflux stimulated by redox activity is independent of plasma membrane ATPase activity

Proton efflux from mesophyll cells of Asparagus sprengeri Regel was inhibited completely by diethylstilbestrol (DES) and NN'-dicyclohexylcarbodiimide (DCCD), known inhibitors of the plasma membrane ATPase. At the concentrations of inhibitors employed, fusicoccin did not reactivate proton efflux. Subsequent addition of ferricyanide however resulted in significant rates of acidification of the medium and reduction of ferricyanide. Similar results were obtained in the light and in the dark. Thus, medium acidification in response to redox activity appears to be independent of the ATP-dependent acidification process.     

Influence of Temperature on Proton Secretion and Hexacyanoferrate (III) Reduction of Zea mays L. Roots

Responses of potassium hexacyanoferrate (III) [HCF(III)] reduction and net proton secretion by Zea mays L. cv Goldprinz roots to changes in ambient temperature were investigated. Arrhenius plots of proton secretion and redox activity showed a constant slope between 5 and 20[deg]C, indicating that reaction kinetics do not change. Proton secretion without HCF(III) was strongly temperature dependent. This dependence was not altered when H+ efflux was stimulated by fusicoccin or by increased K+ concentration. The temperature coefficient for HCF(III) reduction was low, indicating that the velocity of this reaction was limited by apoplastic diffusion of the ferric complex. In the presence of HCF(III) but not hexacyanoferrate (II), temperature dependence of proton efflux markedly declined, indicating fundamental changes in the process(es) contributing to net proton secretion. It is concluded that HCF(III) establishes a proton extrusion path that is directly linked with the reduction reaction.     

Inhibition by fusicoccin of germination of pea seeds

Fusicoccin inhibits the germination of pea (Pisum sativum L. cv Progress 9) seeds by decreasing the growth of the embryonal axis and by stimulating the fresh weight increase of the cotyledons. The growth of isolated embryonal axes in the presence of sucrose and KCl is stimulated by fusicoccin. The effect of fusicoccin on the seeds is not counteracted by sucrose and KCl. Fusicoccin promotes preferentially in the cotyledons a hyperpolarization of the transmembrane electric potential and an increase in the uptake capacity, suggesting the reinforcement of the sink strength of the cotyledons in comparison with the one of the embryonal axis and therefore the inhibition of translocation from the cotyledons of some substance necessary for the growth of the embryonal axis.     

Induction of elongation in maize coleoptiles by hexachloroiridate and its interrelation with auxin and fusicoccin action

The demonstration of an auxin-stimulated NADH-oxidase in the plasma membrane (Brightman et al. 1988. Plant Physiol. 86: 1264–1269) has led to the suggestion that the plasma membrane redox system is involved in the mechanism of auxin action. To evaluate the relevance of this concept in vivo, the influence of micromolar concentrations of hexachloroiridate (IV), an impermeable electron acceptor for the plant plasma membrane redox system, on elongation growth of excised, abraded maize coleoptile ( Zea mays L. cv. Golden Bantam) segments was studied. It was found that the substance induced a rapid growth response if the experiment was carried out in an unbuffered solution. This effect was entirely prevented by a 2 m M phosphate buffer. Nevertheless, the acid-growth-theory does not seem sufficient to explain this effect, since proton extrusion is induced without a lag, whereas increased growth rates commence after a lag phase of 40 min.
If growth is stimulated by a pretreatment with fusicoccin or auxin, hexachloroiridate IV transiently inhibits growth. The kinetics of the response are then determined by the concentrations of hexachloroiridate and auxin or fusicoccin. These results are compatible with the view that the plasma membrane redox system is somehow involved in the control of elongation growth.     

Action of proline on stomata differs from that of abscisic Acid, g-substances, or methyl jasmonate

Methyl jasmonate (MJ) and a mixture of G₁, G₂, and G₃ (G-substances) inhibited stomatal opening in abaxial epidermis of Commelina benghalensis and complete closure occurred at 10⁻⁶ molar MJ, or 10⁻³ molar G-substances compared to 10⁻⁵ molar abscisic acid (ABA). Proline, even at 10⁻³ molar caused only a partial stomatal closure. Apart from ABA, other endogenous plant growth regulators do regulate stomata. Reduction in the stimulation by fusicoccin and complete stomatal closure, at 30 millimolar KCl or less, were affected by ABA, MJ, or G-substances, but not by proline. The action of MJ or G-substances was similar to ABA in decreasing proton efflux and the levels of potassium, malate, or reducing sugars. Proline, however, interfered with starch-sugar interconversion but had no effect on proton efflux or potassium content of epidermis.     

Characteristics and implications of prolonged fusicoccin-induced growth of Avena coleoptile sections

A study has been made of the prolonged growth of Avena coleoptile sections in response to fusicoccin (FC), a phytotoxin that promotes apoplastic acidification. The final amount of FC-induced growth is a function of the FC concentration. Removal of the epidermis speeds up the initial rate of elongation and shortens the duration of the response, without affecting the total amount of extension. A suboptimal FC concentration (7×10⁻⁸ M ) which induces the same rate of proton excretion as does optimal indoleacetic acid (IAA) (1×10⁻⁵ M ), causes elongation which is 60–75% of that induced by IAA in 4 h or 50–65% in 7 h. This suggests that acid-induced extension could make a major contribution to auxin-induced growth for at least 7 h.     

Relationship between ATP Level and Activity of Fusicoccin-stimulated H/K-Exchange System in Plant Tissues

The treatment with fusicoccin causes a slight but significant decrease (about 15%) in the ATP level in pea-internode and maize-coleoptile segments. This decrease is detectable within 15 minutes and is accompanied by a parallel increase in O₂ uptake. Sodium azide inhibits O₂ uptake and completely blocks the stimulation of O₂ uptake by fusicoccin in both pea and coleoptile segments. Benzohydroxamic acid does not affect either basal or fusicoccin-induced O₂ uptake in maize-coleoptile sections. The drop of ATP level induced by various treatments (sodium arsenate, 2-deoxyglucose, limiting O₂, and anaerobiosis) is accompanied by a parallel inhibition of K⁺ uptake in maize coleoptiles treated with or without fusicoccin. These results are consistent with the hypothesis that ATP is the energy source for the fusicoccin-activated H⁺/K⁺-exchange system.     

Promoting effect of fusicoccin on Na+ efflux in barley roots: evidence for a Na+−H+ antiport

Abstract. The effect of fusicoccin (FC) on the K⁺stimulated Na⁺ efflux in root cells of Na⁺ loaded barley roots was studied. FC (0.02 mM) stimulated Na⁺ efflux in the presence of K⁺ and its effect was synergistic with that of K⁺, in a similar way as its effect on proton extrusion. Decreasing the pH of the elution medium promoted Na⁺ efflux and partially replaced the effect of FC. As FC is known to increase the electrochemical proton gradient at the plasmalemma level, these results are consistent with the hypothesis that Na⁺ is extruded in exchange for H⁺. A further support to this view came from the finding that Na⁺ efflux was also promoted by a lipophilic cation, tributylbenzylammonium (TBBA ⁺), which stimulates H ⁺ extrusion and is generally accepted not to enter the cells by means of the same carrier as K ⁺.     

Early elicitor-induced events in chickpea cells: functional links between oxidative burst, sequential occurrence of extracellular alkalinisation and acidification, K+/H+ exchange and defence-related gene activation

Elicitation of cultured chickpea (Cicer arietinum L.) cells stimulates a signal transduction pathway leading to several rapid responses: (1) oxidative burst, (2) extracellular alkalinisation, (3) extracellular acidification, (4) transient K+ efflux, and (5) activation of defence related genes all within 2 hours. Induced genes are encoding acidic and basic chitinases, a thaumatin-like protein and isoflavone reductase. All these elicitor-induced responses are inhibited by the Ser/Thr protein kinase inhibitor staurosporine and the anion channel blocker anthracene-9-carboxylic acid but stimulated by the Ser/Thr protein phosphatase 2A inhibitor cantharidin. The oxidative burst leads to a transient extracellular H2O2 accumulation which seems to be preceded by O2- production, indicating dismutation of O2- to H2O2. The oxidative burst is accompanied by transient alkalinisation of the culture medium which is followed by long-lasting extracellular acidification. An 80 percent inhibition of the alkalinisation after complete inhibition of the H2O2 burst with diphenylene iodonium indicates that the elicitor induced increase of extracellular pH is mainly based on a proton consumption for O2-dismutation. A simultaneous deactivation of the plasma membrane H+-ATPase during oxidative burst and extracellular alkalinisation is also suggested. The elicitor-stimulated extracellular acidification is inhibited by the plasma membrane H+-ATPase inhibitor N, N'-dicyclohexylcarbodiimide assuming a reactivation of the H+-ATPase 25 min after elicitation. Extracellular acidification seems not to be necessary for elicitor-induced activation of defence related genes. Opposite modulation of K+ and proton fluxes after elicitation and/or treatment with the H+-ATPase effectors fusicoccin or N, N'-dicyclohexylcarbodiimide indicate that the elicitor induced transient K+ efflux is regulated by a K+/H+ exchange reaction.     

Carriers for abscisic acid and indole-3-acetic acid in primary roots: their regional localisation and thermodynamic driving forces

A carrier for the uptake of abscisic acid (ABA) is present in the tips and elongating zones of primary roots of both leguminous (runner bean, French bean, pea) and non-leguminous (sunflower, maize) seedlings. No ABA carrier was present in more mature root regions. For indole-3-acetic acid both carrier-mediated uptake and a 2,3,5-triiodobenzoate-sensitive efflux component are present in growing and in non-elongating runner-bean root tissues. Both ABA and indole-3-acetic acid carriers were inactivated by protein-modifying reagents. The driving forces for the carrier systems were studied using reagents, (KCl, fusicoccin, vanadate, dicyclohexylcarbodiimide, proton ionophores and azide) known to modify transmembrane pH (ΔpH) and electricla gradients (ΔE) and whose effects were independently monitored using radiolabelled, lipophilic, weak acids as probes. For abscisic acid the carrier-mediated uptake depend on ΔpH and the nonsaturable component of uptake, due to diffusion of undissociated ABA. The maximum velocity of the carrier is greater at pH 4 than at pH 5, although the Michaelis constants are similar. Modification of ΔE did not alter ABA net uptake but effects on the indole-3-acetic acid system consistent with perturbation of an electrogenic 2,3,5-triiodobenzoate-sensitive component were observed. It is suggested that the ABA carrier is an ABA anion/hydrogen ion symport or, less likely, represents facilitated diffusion of undissociated ABA.     

INTERACTING ROLES OF GIBBERELLIN AND ETHYLENE IN COROLLA EXPANSION OF IPOMOEA NIL (CONVOLVULACEAE)

The relatively rapid and extensive corolla expansion in Ipomoea nil (L.) Roth cv. ‘Scarlett O'Hara’ appears to be initiated by a shift in the balance between a growth promoter (gibberellin, GA) and a growth inhibitor (ethylene) two days before anthesis. The effects of applied growth regulators in vitro, were measured as a change in area of segments from whole young corollas 16–17 mm long. Applied gibberellic acid (GA₃) strongly promoted growth, while a GA action inhibitor (ancymidol) reduced growth. Inhibitors of GA biosynthesis (AMO-1618, paclobutrazol, tetcyclasis, and chlorocholine chloride) had little effect, implying that the GAs were not being synthesized within the corolla segments, at least at this stage of corolla growth. Both the level of endogenous GAs and response of the segments to sucrose increased as the corolla size also increased from 15 to 20 mm in length. Applied 1-aminocyclopropane-l-carboxylic acid (ACC), an ethylene precursor, inhibited the response of corollas to applied GA₃. Applications of ethylene biosynthesis inhibitors (AVG and cobalt ions) promoted growth of the corolla segments. Rapid ethylene production by corollas 15–17 mm long (48 hr before flower opening) appeared to inhibit the growth that may have been induced by low levels of endogenous GAs. Older corollas (longer than 18 mm) had very low levels of ethylene production and much higher levels of GA-like substances; these changes apparently allow the corollas to begin to expand in vivo and to strongly respond to sucrose applications alone in vitro. Acid-induced growth does not seem to be an important component of corolla expansion; applied fusicoccin (a proton efflux promoter) and sodium orthovanadate (a proton efflux inhibitor) had no significant effect on growth of the 16–17 mm long corollas. Buffers (Good's and phosphate) over a wide pH range had no effect on corolla expansion. Taken together, our results indicate that the regulation of corolla expansion depends (at least in part) on compensatory shifts in the levels of two plant growth regulators [PGRs], ethylene and GA.     

Light-promoted diffusional amino acid efflux from Commelina leaf disks

The release (=the measured loss) of amino acids was studied in Commelina benghalensis leaf disks. The release is assumed to be the result of influx and efflux, therefore, both movements were investigated.The uptake of ¹⁴C-labeled valine exhibited a biphasic isotherm. The uptake was pH-dependent, especially at low substrate concentrations (pH optimum 4.8). Signals for amino acid/proton co-transport were observed: stimulation of the uptake by fusicoccin (FC), inhibition by diethylstilbestrol (DES) or by high K⁺ concentrations. In the light, the ATP level of the disks was maintained during the uptake period (2 h), in darkness the ATP content decreased from 87 to 24 nmol g^–1 fr. wt. However, light-promoted uptake, which is explained in the proton pump concept by an intensified proton extrusion as the result of high ATP production, was lacking.The release of amino acids was increased by washing with p-chloromercuriphenyl sulphonic acid (PCMBS), nystatin, 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), or KCN. The release (Q₁₀ about 1.5) was independent of the external pH and was linearly related to the intracellular amino acid concentration. Light enhanced the rate of release to the same extent at all intracellular concentrations. The present results suggest that the release is balanced by a, at least partially, proton-driven influx and a diffusional ligh-promoted efflux. A provisional model shows how the diffusional effulx can be indirectly controlled by a counter-flow fueled by the metabolism.Abbreviations PCMBS p-chloromercuriphenylsulphonic acid - CCCP carbonyl cyanide m-chlorophenyl hydrazone - DES diethylstibestrol - DCMU 3 (3,4-dichlorophenyl)-1, 1-dimethyl urea - TRIS 2-amino-2-(hydromethyl)propane-1,3 diol - MES 2-(N¹-morpholino) ethane sulphonic acid monohydrate - FC fusicoccin