Auxin-induced elongation growth and expressions of cell wall-bound exo- and endo-beta-glucanases in barley coleoptiles

When auxin stimulates rapid cell elongation growth of cereal coleoptiles, it causes a degradation of 1,3:1,4-beta-glucan in hemicellulosic polysaccharides. We examined gene expressions of endo-1,3:1,4-beta-glucanase (EI) and exo-beta-glucanase (ExoII), of which optimum pH are about 5, and molecular distribution of hemicellulosic polysaccharides in barley (Hordeum vulgare L.) coleoptile segments treated with or without IAA. IAA (10(-5) M) stimulated the gene expression of EI, while it did not affect that of ExoII. IAA induced gene expression of EI after 4 h and increased wall-bound glucanase activity after 8 h. The molecular weight distribution of hemicellulosic polysaccharides from coleoptile cell walls was shifted to lower molecular weight region by 2 h of IAA treatment. Fusicoccin (10(-6) M) mimicked IAA-induced elongation growth and the decrease in molecular weight of hemicellulosic 1,3:1,4-beta-glucan of coleoptiles in the first 4 h, but it did not promote elongation growth thereafter. These facts suggest that acidification of barley cell walls by IAA action enhances pre-existing cell wall-bound glucanase activity in the early first phase of IAA-induced growth and the late second phase involves the gene expression of EI by IAA.     

Effect of temperature on the dose–response curves for auxin-induced elongation growth in maize coleoptile segments

The dose–response curves for IAA-induced growth in maize coleoptile segments were studied as a function of time and temperature. In addition, the kinetics of growth rate responses at some auxin concentrations and temperatures was also compared. It was found that the dose–response curves for IAA-induced elongation growth were, independently of time and temperature, bell-shaped with an optimal concentration at 10⁻⁵ M IAA. The kinetics of IAA-induced growth rate responses depended on IAA concentration and temperature, and could be separated into two phases (biphasic reaction). The first phase (very rapid) was followed by a long lasting one (second phase), which began about 30 min after auxin addition. For coleoptile segments incubated at 30°C, the amplitudes of the first and second phase were significantly higher, when compared with 25°C, at all IAA concentrations studied. However, when coleoptile segments were incubated at 20°C, the elongation growth of coleoptile segments treated with suboptimal IAA concentrations was diminished, mainly as a result of both phases reduction. In conclusion, we propose that the shape of the dose–response curves for IAA-induced growth in maize coleoptile segments is connected with biphasic kinetic of growth rate response.     

Inside or outside? Localization of the receptor relevant to auxin-induced growth

The localization of the auxin receptor relevant to the control of elongation growth is still a matter of controversy. Auxin-induced elongation of maize coleoptile segments was measured by means of a high resolution auxanometer. When indole-3-acetic acid (IAA) was removed from the bathing solution, a rapid cessation of auxin-induced elongation was detected. This decline was delayed when the auxin efflux carrier was blocked by the phytotropins naphthylphthalamic acid (NPA) and pyrenoylbenzoic acid (PBA) or by triiodobenzoic acid (TIBA). The IAA concentration in NPA-pretreated segments was 2–3 times higher than in NPA-free controls 35 min after the removal of IAA in the bathing medium.
A similar rapid drop of growth after removal of auxin was observed for the rapidly-transported synthetic auxin, naphthaleneacetic acid (NAA). When the auxin efflux was blocked, growth induced by NAA was sustained much longer than IAA-stimulated elongation.
In comparison with NAA, the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) is known to be excreted very slowly by the efflux carrier. 2,4-D-induced growth remained at a stimulated level when the auxin was washed off, even in the absence of any auxin efflux inhibitor. We conclude from these results that the presence of intracellular auxin is a necessary and sufficient condition for sustained auxin-induced elongation growth, at least for the phases during the 2 h after its application. Consequently, we postulate the existence of an intracellular auxin receptor relevant to the control of growth.     

Auxin, actin and growth of the Arabidopsis thaliana primary root

To understand how auxin regulates root growth, we quantified cell division and elemental elongation, and examined actin organization in the primary root of Arabidopsis thaliana. In treatments for 48 h that inhibited root elongation rate by 50%, we find that auxins and auxin-transport inhibitors can be divided into two classes based on their effects on cell division, elongation and actin organization. Indole acetic acid (IAA), 1-naphthalene acetic acid (NAA) and tri-iodobenzoic acid (TIBA) inhibit root growth primarily through reducing the length of the growth zone rather than the maximal rate of elemental elongation and they do not reduce cell production rate. These three compounds have little effect on the extent of filamentous actin, as imaged in living cells or by chemical fixation and immuno-cytochemistry, but tend to increase actin bundling. In contrast, 2,4-dichlorophenoxy-acetic acid (2,4-D) and naphthylphthalamic acid (NPA) inhibit root growth primarily by reducing cell production rate. These compounds remove actin and slow down cytoplasmic streaming, but do not lead to mislocalization of the auxin-efflux proteins, PIN1 or PIN2. The effects of 2,4-D and NPA were mimicked by the actin inhibitor, latrunculin B. The effects of these compounds on actin were also elicited by a 2 h treatment at higher concentration but were not seen in two mutants, eir1-1 and aux1-7, with deficient auxin transport. Our results show that IAA regulates the size of the root elongation zone whereas 2,4-D affects cell production and actin-dependent processes; and, further, that elemental elongation and localization of PINs are appreciably independent of actin.     

Evidence against the acid-growth theory of auxin action

Four experimental predictions of the acid-growth theory of auxin (indole-3-acetic acid, IAA) action in inducing cell elongation were reinvestigated using abraded segments of maize (Zea mays L.) coleoptiles. i) Quantitative comparison of segment elongation and medium-acidification kinetics measured in the same sample of tissue reveals that these IAA-induced processes are neither correlated in time nor responding coordinately to cations present in the medium. ii) Exogenous protons are not able to substitute for IAA in causing segment elongation at the predicted pH of 4.5–5.0. Instead, external buffers induce significant segment elongation only below pH 4.5, reaching a maximal response at pH 1.75–2.5. Acid and IAA coact additively, and therefore independently, in the whole range of feasible pH values. iii) Neutral or alkaline buffers (pH 6–10) are unable to abolish the IAA-mediated growth response and have no effect on its lag-phase. iv) Fusicoccin, at a concentration producing the same H⁺ excretion as high concentrations of IAA, is ineffective in inducing segment elongation. Moreover, sucrose and other sugars can quantiatively substritute for IAA in inducing H⁺ excretion but are likewise ineffective in inducing elongation. It is concluded that these results are incompatible with the acid-growth theory of auxin action.Abbreviations IAA indole-3-acetic acid - FC fusicoccin     

Physiological evidence that the primary site of auxin action in maize coleoptiles is an intracellular site

To locate functionally the primary site of auxin action in growing cells, the pool of auxin relevant to induction of growth in maize (Zea mays L.) coleoptile sections was determined. A positive correlation was consistently noted between growth and intracellular levels of indole-3-acetic acid (IAA), i.e. growth appears to be relatively independent of the external level of IAA. N-1-Naphthylphthalamic acid (NPA), a potent inhibitor of auxin transport, was used to enhance accumulation of IAA in coleoptile cells. From the use of NPA, it is shown that: 1) increasing the accumulation of IAA in cells, while the external concentration is held constant, resulted in a concomitant increase in growth, and 2) blocking the exit of IAA from cells with NPA sustained an IAA-induced growth response in the absence of externally applied IAA. Furthermore, the absence of any alterations in auxin binding to microsomal fractions by NPA indicates that the action of NPA in causing enhancement of auxin-induced growth is based upon its inhibition of efflux of IAA from the cells. This research was supported by National Science Foundation grant No. DMB 8515925. The careful assistance of Laurie Brulport is gratefully acknowledged.     

Auxin-stimulated ATPase in membrane fractions from pumpkin hypocotyls (Cucurbita maxima L.)

Membrane fractions from Cucurbita maxima hypocotyls were isolated in a medium which inhibits the action of endogenous phospholipases. After removal of soluble phosphatases by Sepharose 2B-CL column chromatography, an auxin-stimulated ATPase activity was found in membrane fractions from linear sucrose gradients. In the presence of 10^-4 M phenylacetic acid (PAA), the stimulation by indol-3-acetic acid (IAA) exhibited a bimodal concentration dependence with maximal stimulation of about 50% at 10^-6 M IAA. Without PAA, only a high concentration of 10^-4 M IAA was stimulatory, whereas 10^-6 M IAA had no apparent effect and 10^-8 M IAA exhibited weak inhibition. PAA alone had only weak or no effects. The effects of IAA must be considered as hormone-specific. The ATPase activity in the presence of 10^-4 M PAA was activated only by 2,4-dichlorophenoxyacetic acid (2,4-D), an active auxin analogue, but not by the inactive stereoisomers, 2,3-D and 3,5-D. Comparison with marker enzyme profiles suggested that part of the auxin-stimulated ATPase was localized on plasma membranes as well as other compartments. Thus, the auxin-stimulated ATPase may become a useful tool in the investigation of the mechanism of action of auxin.Abbrevations 2,4-D 2,4-dichlorophenoxyacetic acid - 2,3-D 2,3-dichlorophenoxyacetic acid - 3,5-D 3,5-dichlorophenoxyacetic acid - IAA indol-3-acetic acid - PAA phenylacetic acid - MES (2-(N-morpholino))-ethanesulfonic acid - EDTA ethylenediamine tetraacetic acid     

Rapid-growth responses of corn root segments: Effect of auxin on elongation

The effect of indole-3-acetic acid (IAA) on the elongation rates of 2 mm corn (Zea mays L.) root segments induced by citrate-phosphate buffer (or unbuffered) solutions of pH 4.0 and 7.0 was studied. At pH 7.0, auxin initially reduced the elongation rate in both buffered and unbuffered solutions. Only in buffer at pH 7.0 was auxin at a concentration of 0.1 M found to promote the elongation rate though briefly. THis promoted rate represented only ca. 20% of the rate achieved with only buffer at pH 4.0. Auxin in pH 4.0 buffered and unbuffered solutions only served to reduce the elongation rates of root segments. Some comparative experiments were done using 2 mm corn coleoptile segments. Auxin (pH 6.8) promoted the elongation rate of coleoptile segments to a level equal or greater than the maximal H ion-induced rate. The two responses of root segments to auxin are compared to auxin action in coleoptile growth.     

Influence of various media constituents on the growth of Cinchona ledgeriana tissue cultures and the production of alkaloids and anthraquinones therein

Using the methods reported by De Fossard et al. (11) the influence of various media constituents on the growth and the alkaloid and anthraquinone production in Cinchona ledgeriana callus cultures was studied. Growth and indole alkaloid production (e.g. cinchonamine) was improved by higher auxin levels. The best growth was observed in the light, although many media resulted in no growth at all in the light. Anthraquinone production was highest at lower auxin levels. Quinoline alkaloid levels (e.g. quinidine) were highest in media with low auxin concentrations. Low and medium cytokinin concentration benefited the quinoline alkaloid production.From the results it was concluded that the pathways leading to the various secondary products, anthraquinones, indole alkaloids and quinoline alkaloids are, at least partly, regulated independently.Abbreviations used IAA indol-acetic acid - IBA indol-butyric acid - NAA -naphtaleneacetic acid - NOA 2-naphtoxy-acetic acid - 2,4-D 2,4-dichlorophenoxy-acetic acid - pCPA parachlorophenoxy-acetic acid - BA benzyladenine     

The effects of electric field on the growth of intact seedlings and coleoptile segments ofZea mays L.

The experiments were carried out with 96-h-old intact maize seedlings and 10 mm long coleoptile segments cut 4 mm below the tip. The electric fields were applied longitudinally along the seedlings. The electric field (15 V) caused inhibition of the elongation growth of intact seedlings which was dependent on both the polarity and the duration of the applied voltage. The growth inhibition was greater when the tip of the shoot was positive relative to the roots. The electric field also caused inhibition of indole-3-acetic acid (IAA) and fusicoccin (FC) induced growth of maize coleoptile segments excised from electrically treated seedlings. IAA-induced growth of coleoptile segments was greater when the tip of the shoot was negative to the roots (not in the case of FC-treated segments and intact seedlings). It was suggested that apart from the changes induced by electric field in transport system of auxin the electric field affected also the activity of plasmalemma proton pump.     

Auxin carriers in Cucurbita vesicles

Carrier-mediated uptake of indole-3-acetic acid (IAA) by microsomal vesicles from Cucurbita pepo L. hypocotyls was strongly inhibited by 2,4-dichlorophenoxyacetic acid (2,4-D; i ₅₀= 0.3 M) but only weakly by 1-naphthylacetic acid (NAA). The fully ionised auxin indol-3-yl methanesulphonic acid also inhibited (i ₅₀=3 M). The same affinity ranking of these auxins for the uptake carrier, an electroimpelled auxin anion-H⁺ symport, is demonstrable in hypocotyl segments. The specificity of the auxin-anion eflux carrier was tested by the ability of different nonradioactive auxins to compete with [³H]IAA and reduce the stimulation of net radioactive uptake by N-1-naphthylphthalamic acid (NPA), a noncompetitive inhibitor of this carrier. By this criterion, NAA and IAA had comparable affinities, with 2,4-D interaction more weakly. Stimulation of [³H]IAA uptake by NAA, as a result of competition for the efflux carrier, could also be demonstrated when a suitable concentration of 2,4-D was used selectively to inhibit the uptake carrier. However, when [³H]NAA was used, no stimulation of its association with vesicles by NPA, 2,3,5-triiodobenzoic acid, or nonradioactive NAA was found. In hypocotyl segments, [³H]NAA net uptake was much less sensitive to NPA stimulation than was [¹⁴C]IAA uptake. The apparent contradictions concerning NAA could be explained by carrier-mediated auxin efflux making a smaller relative contribution to the overall transport of NAA than of IAA. The relationship between carrier specificity as manifested in vitro and the specificity of polar auxin transport is discussed.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - ION3 mixture of 4 M carbonylcyanide m-chlorophenylhydrazone, nigericin and valinomycin - IMS indol-3-yl methanesulphonic acid - NAA 1-naphthylacetic aci - NPA N-1-naphthylphthalamic acid     

Stimulatory effects of potassium iodide on auxin action in the coleoptile segments of maize (Zea mays)

Potassium iodide (KI) was found to stimulate IAA-induced elongation of coleoptile segments in maize (Zea mays L.). The promoting effects of KI on coleoptile elongation, which were optimal at 1 mM in the presence of IAA, did not occur as a result of better conservation of IAA in the incubation medium. In addition, KI did not affect fusicoccin- or epibrassinolide-induced elongation. Additionally, sodium iodide (NaI) induced similar stimulatory effects on IAA-induced elongation, however, potassium chloride (KCl) showed no effect, suggesting that iodide is the active component. KI also enhanced IAA-induced ethylene biosynthesis in maize coleoptile segments. Taken together, these results suggest the involvement of KI-sensitive step(s) in auxin action before effectors of the signal transduction pathway split to elongation growth and ethylene biosynthesis. In-yong Hwang and Soo Chul Chang contributed equally to this work.     

The effects of 3,5-diiodo-4-hydroxybenzoic acid on the oxidation of IAA and auxin-induced ethylene production by cress root segments

Summary In previous research here, 3,5-diiodo-4-hydroxybenzoic acid (DIHB) was shown to promote the elongation of roots of cress (Lepidium sativum) seedlings growing in light, and to inhibit the auxin-induced production of ethylene in this tissue. Although DIHB is a cofactor for the oxidation of indole-3-acetic acid (IAA) by horse-radish peroxidase, it inhibits the decarboxylation of [1-¹⁴C]IAA by segments excised from cress roots. The inhibition by DIHB of ethylene production by this tissue does not, therefore, arise from a reduction of IAA levels. These findings are discussed in relation to the effects of DIHB on cress root growth.Abbreviations IAA indole-3-acetic acid - DIHB 3,5-diiodo-4-hydroxybenzoic acid - DCP 2,4-dichlorophenol - 2,4-D 2,4-dichlorophenoxyacetic acid This study forms part of a research project to be submitted by M.L.R. for PhD degree and supported by a grant from Consejo Nacional de Ciencia y Tecnología (México).     

Effect of Peeling on IAA-induced Growth in Avena Coleoptiles

The act of peeling removes the epidermis exclusively from Avenacoleoptiles. Peeling inhibits IAA-induced growth, by inhibitingthe growth of segments incubated in the presence of IAA, andpromoting that of those incubated in water. The magnitude ofthe inhibition of IAA-induced growth is proportional to theamount of epidermis removed. It is shown that neither lateralswelling, wounding, anaerobiosis, nor exposure to supraoptimalconcentrations of IAA cause the inhibition. It is concludedthat in Avena coleoptiles the epidermis regulates the rate ofexpansion of the underlying parenchyma cells and is the principaltarget of IAA-action. Avena sativa L., oat, coleoptile, indol-3-ylacetic acid, auxin, extension growth     

Role of Auxin in Growth of Inhibitor-treated Oat Coleoptile Tissue

The role of auxin in the recovery of plant tissue from oxidant treatment was investigated. Treatment of oat coleoptile sections with concentrations of indoleacetic acid (IAA) or 2,4-dichlorophenoxyacetic acid (2,4-D) optimal for normal growth, following pretreatment with moderately inhibiting levels of peroxyacetyl nitrate (PAN) immediately accelerated recovery of growth rate. In some cases inhibition was also less at supraoptimal values of auxin. Treatment of ozonepretreated tissue with IAA or 2,4-D enhanced inhibition at high levels of auxin and produced an optimal growth concentration level which was lower than for sections not given ozone pretreatment. Auxin treatment also reduced the degree of inhibition in fluoride and iodoacetamide-pretreated sections. Mechanisms by which auxin-induced recovery from inhibition may occur are discussed.     

Auxin binding in roots: A comparison between maize roots and coleoptiles

Auxin binding onto membrane fractions of primary roots of maize seedlings has been demonstrated using naphth-1yl-acetic acid (NAA) and indol-3yl-acetic acid (IAA) as ligands. This binding is compared with the already well characterized interaction between auxins and coleoptile membranes. The results indicate that while kinetic parameters are of the same order for root and coleoptile binding, a number of differences occur with respect to location in cells and relative affinity. The possible significance of the existence of such binding sites in root cells is discussed in relation to auxin action.Abbreviations 4-Cl-PA 4-chlorophenoxyacetic acid - EDTA ethylene diamine tetracetic acid - IAA indol-3yl-acetic acid - MCPA 2-methyl-4-chlorophenoxyacetic acid - NAA naphth-1yl-acetic acid - 2-NAA naphth-2yl-acetic acid - Tris 2-amino-2-(hydroxymethyl) propane-1,3 diol - TIBA 2,3,5 triiodobenzoic acid - NPA naphthylphthalamic acid - PCIB 4-chlorophenoxyisobutyric acid - PCPP 4-chlorophenoxyisopropionic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

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     

Further biological characteristics of galactoglucomannan oligosaccharides

The biological activity of cell wall-derived galactoglucomannan oligosaccharides (GGMOs) was dependent on their chemical structure. Galactosyl side chains linked to the glucomanno-core influenced their inhibition of elongation growth of pea (Pisum sativum L. cv. Tyrkys) stem segments induced by 2,4-dichlorophenoxyacetic acid (2,4-D). Reduction of the number of galactosyl side chains in GGMOs caused stimulation of the endogenous growth. Modification on the glucomanno-reducing end did not affect significantly the activity of these oligosaccharides. GGMOs inhibited also the elongation induced by indole-3-acetic acid (IAA) and gibberellic acid (GA₃). In the presence of IAA the elongation growth was inhibited to 20 – 35 % after 24 h of incubation depending on GGMOs concentrations (1 μM, 10 nM, 0.1 nM), similarly as in the presence of 2,4-D, which confirms the hypothesis of GGMOs antiauxin properties. The elongation induced by GA₃ was inhibited to 25 – 60 %, however, the time course of inhibition was different compared with IAA and 2,4-D. The highest inhibition was determined already after 6 h of incubation with a significant decrease after this time. The results indicated a competition between GGMOs and growth regulators.     

Hexacyanoferrate (III) stimulation of elongation in coleoptile segments fromZea mays L.

Summary The influence of exogenous potassium hexacyanoferrate (III) (HCF III) on elongation of maize (Zea mays L.) coleoptile segments was investigated. Addition of HCF III led to a strong stimulation of growth both in the presence and absence of indole-3-acetic acid (IAA). The magnitude of growth stimulation was dependent on the presence of IAA, HCF III concentration, incubation time, and phase growth. The reduced form, potassium hexacyanoferrate (II), was without effect on growth. In the presence of HCF III, elongation was suppressed when coleoptile segments were treated with N,N-dicyclohexylcarbodiimide, cycloheximide or atebrine (quinacrine). The addition of HCF III stimulated the IAA-induced proton extrusion, and the e^–/H⁺ ratio decreased with incubation time. HCF III also strongly stimulated elongation ofAvena saliva L. coleoptile segments andGlycine max L. hypocotyl segments. These results suggested that a plasma membrane redox system (NADH oxidase type I) may be involved in the regulation of growth through the activity of the plasma membrane-bound ATPase.Abbreviations CH cycloheximide - DCCD N,N-dicyclohexylcarbodiimide - HCF III potassium hexacyanoferrate (III) (potassium ferricyanide) - HCF II potassium hexacyanoferrate (II) (potassium ferrocyanide) - IAA indole-3-acetic acid     

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^-5moll^-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.Abbreviations IAA indoleacetic acid - GA₃ gibberellic acid - FC fusicoccin - PD electric potential difference between the vacuole and the external medium