Transmembrane electrical potentials in growing maize roots

The anti-auxin 4-chlorophenoxyisobutyric acid (PCIB) applied at a concentration of 10^-2 mol m^-3 to maize root segments was found to induce a transmembrane electrical potential of up to-130 mV (pd of 30 mV). The kinetics of this response were comparable to the time scale for PCIB-stimulated H⁺-extrusion. Both effects are eliminated by the addition of p-fluoromethoxycarbonyl cyanide phenylhydrazone (FCCP). Treatment with fusicoccin (FC) and PCIB together does not result in a hyperpolarization greater than with FC alone. Benzoic acid (10^-2 mol m^-3) had no effect on the transmembrane electrical potentials. These results are discussed in relation to a possible electrogenic proton pump which may be regulated by perturbations in the cellular auxin content or activity.Abbreviations ATPase adenosine triphosphatase - FC fusicoccin - FCCP p-fluoromethoxy carbonylcyanide phenylhydrazone - IAA indole-3yl-acetic acid - NAA naphthyl-lylacetic acid - PCIB 4-chlorophenoxyisobutyric acid - PD potential difference     

Energetics of threonine uptake by pod wall tissues of Vicia faba L.

Kjeldahl assays showed that the pod wall of Vicia faba fruits behaves as a transitory reservoir of nitrogen. We have studied the properties and energetics of amino-acid uptake during the accumulating stage of pod wall development. A comparative analysis using various inhibitors or activators of the proton pump has been carried out i) on threonine uptake, ii) on the acidifying activity of the tissues, and iii) on the transmembrane potential difference of mesocarp cells. Except for the effect of dicyclohexylcarbodiimide which could not be satisfactorily explained, all other results obtained with ATPase inhibitors, uncouplers and fusicoccin were consistent with the view of a transport energized by the proton-motive force. Adding threonine to a medium containing fragments of pericarp or of endocarp induced a pH change (to-wards more alkaline values) of the medium and a membrane depolarization of the storage cells which depended on the amino-acid concentration added. These data indicate H⁺-threonine cotransport in the pod wall of broad bean. Moreover, because p-chloromercuribenzenesulphonic acid inhibits threonine uptake without affecting the transmembrane potential difference, it is concluded that the threonine carrier possesses a functional SH-group located at the external side of the plasmalemma.Abbreviations CCCP carbonylcyanide- m-chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DES diethylstilbestrol - DNP 2,4-dinitrophenol - FC fusicoccin - PCMBS p-chloromercuribenzenesulphonic acid - PD potential difference     

Effects of fusicoccin and abscisic acid on glucose uptake into isolated beetroot protoplasts

Uptake of glucose, 3-O-methylglucose and sucrose into beetroot protoplasts is considerably stimulated by 10^–6M fusicoccin. This effect is decreased in the presence of 10mM Na⁺ or K⁺, 2 mM Mg²⁺ or Ca²⁺. Whereas fusicoccin causes no change in the pH-optimum of the sugar uptake (pH 5.0), the apparent K_m of this uptake which obeys a biphasic kinetics is decreased by the action of fusicoccin. In the protoplast suspension, fusicoccin induces an acidification which is suppressed by uncoupling agents. Correspondingly, uncouplers as well as vanadate and diethylstilbestrol markedly inhibit the effect of fusicoccin on sugar uptake. The present data support the view that glucose uptake into beetroot protoplasts depend on the proton-pumping activity of the plasmalemma-ATPase. cis–Abscisic acid diminishes significantly the fusicoccin-enhanced glucose uptake. By using a radioimmunoassay, the internal abscisic acid content of the protoplast was estimated to be in the range of 10^–6 M. Protoplasts isolated from bundle tissue contain twice as much abscisic acid as those derived from storage parenchyma. Because protoplasts from the bundle tissue were shown to take up sugars much faster than those from the storage cells, the observed effect of abscisic acid might reflect an involvement of this hormone in the regulation of carbohydrate partitioning in the beet.Abbreviations ABA cis–abscisic acid - bundle protoplast protoplasts isolated from the conducting tissue of beetroots - DES diethylstilbestrol - FC fusicoccin - 3-OMG 3-O-methylglucopyranose - PCMBS p–chloromercuribenzenesulfonic acid - storage protoplasts protoplasts isolated from storage parenchyma     

Movement and compartmentation of abscisic acid in guard cells of Valerianella locusta: Effects of osmotic stress,external H+-concentration and fusicoccin

Epidermal peels of Valerianella locusta were acid-treated for 1 h at pH 3.9 to kill all cells other than guard cells. These guard-cell preparations were used to explore the steady-state one-way fluxes and the cytoplasmic and vacuolar contents of abscisic acid (ABA). The method of compartmental analysis has been applied. The intracellular ABA concentrations were surprisingly high. At an external pH of 5.8 the cytoplasm contained 1.28 mmol·dm^-3 of ABA, twice of the amount which accumulated in the vacuoles (0.57 mmol·dm^-3). The fluxes of ABA at the plasmalemma (_oc=_oc=0.43 fmol · cell ^–1 · h ^–1) were higher than those at the tonoplast (_cv=_vc=0.12 fmol · cell ^–1 · h ^–1). Moderate stress (0.1 and 0.3 mol·dm^-3 sorbitol in the medium) caused a change in the kinetics of ABA movement. The rate constants of the fluxes from the cytoplasm into the vacuole (_cv) and into the apoplast (_co) were increased while the rate constant of the flux from the vacuoles into the cytoplasm (_vc) was decreased. As a consequence the amount of ABA sequestered in the vacuole remained unchanged; the cytoplasmic ABA content, however, was reduced to only 20% of that found in the control treatments (no sorbitol in the medium). Under moderate stress, one Valerianella guard cell released rapidly about 0.36 fmol·cell^-1 to its direct cell-wall space. This surprising result is discussed in regard to rapid stomatal closure under reduced water supply.Abbreviations ABA abscisic acid - FC fusicoccin     

Changes in abscisic acid and its glucose ester in Phaseolus vulgaris L. during chilling and water stress

Levels of free and conjugated abscisic acid (ABA) were determined in leaves and roots of intact bean (Phaseolus vulgaris L., cv. Mondragone) seedlings under chilling (3C) and drought as well as during recovery from stress. Abscisic acid-glucose ester (ABAGE) was the only conjugate releasing free ABA after alkaline hydrolysis of the crude aqueous extracts. During the first 20–30 h chilled plants rapidly dehydrated and wilted without any change in ABA and ABAGE levels. Subsequently, leaf and root ABA levels increased and plants regained turgor. ABAGE concentration showed a slight increase in leaves but not in roots. Upon recovery from chilling a transient, but significant, rise in leaf ABA content was observed, while no appreciable change in ABAGE was found. Drought triggered ABA accumulation in leaves and roots, while a rise in ABAGE content was detected only in leaf tissues. Recovery from stress caused a drop in ABA levels without a correspondent increase in ABAGE concentration. We conclude that ABAGE is not a source of free ABA during either chilling or water stress and that only a small proportion of the ABA produced under stress is metabolised to ABAGE during recovery.Abbreviations ABA = abscisic acid - ABAGE = abscisic acid-glucose ester - DW = dry weight - FW = fresh weight - RIA = radioimmunoassay - RWC = relative water content - w = water potential - o = osmotic potential - p = turgor potential     

Dormancy termination of western white pine (<Emphasis Type="Italic">Pinus monticola</Emphasis> Dougl. Ex D. Don) seeds is associated with changes in abscisic acid metabolism

Western white pine (Pinus monticola) seeds exhibit deep dormancy at maturity and seed populations require several months of moist chilling to reach their uppermost germination capacities. Abscisic acid (ABA) and its metabolites, phaseic acid (PA), dihydrophaseic acid (DPA), 7-hydroxy ABA (7OH ABA) and ABA-glucose ester (ABA-GE), were quantified in western white pine seeds during dormancy breakage (moist chilling) and germination using an HPLC–tandem mass spectrometry method with multiple reaction monitoring and internal standards incorporating deuterium-labeled analogs. In the seed coat, ABA and metabolite levels were high in dry seeds, but declined precipitously during the pre-moist-chilling water soak to relatively low levels thereafter. In the embryo and megagametophyte, ABA levels decreased significantly during moist chilling, coincident with an increase in the germination capacity of seeds. ABA catabolism occurred via several routes, depending on the stage and the seed tissue. Moist chilling of seeds led to increases in PA and DPA levels in both the embryo and megagametophyte. Within the embryo, 7OH ABA and ABA-GE also accumulated during moist chilling; however, 7OH ABA peaked early in germination. Changes in ABA flux, i.e. shifts in the ratio between biosynthesis and catabolism, occurred at three distinct stages during the transition from dormant seed to seedling. During moist chilling, the relative rate of ABA catabolism exceeded ABA biosynthesis. This trend became even more pronounced during germination, and germination was also accompanied by a decrease in the ABA catabolites DPA and PA, presumably as a result of their further metabolism and/or leaching/transport. The transition from germination to post-germinative growth was accompanied by a shift toward ABA biosynthesis. Dormant imbibed seeds, kept in warm moist conditions for 30 days (after an initial 13 days of soaking), maintained high ABA levels, while the amounts of PA, 7OH ABA, and DPA decreased or remained at steady-state levels. Thus, in the absence of conditions required to break dormancy there were no net changes in ABA biosynthesis and catabolism.Abbreviations ABA abscisic acid - ABA-GE abscisic acid glucose ester - DPA dihydrophaseic acid - 7OH ABA 7-hydroxy abscisic acid - 8OH ABA 8-hydroxy abscisic acid - MRM multiple reaction monitoring - PA phaseic acid     

The effect of Cl- upon the sensitivity of starch-containing and starch-deficient stomata and guard cell protoplasts towards potassium ions,fusicoccin and abscisic acid

Chloride ions are necessary to compensate for the positively charged potassium ions imported into guard cells of Allium cepa L. during stomatal opening. Therefore an external Cl^- supply of intact Allium plants is important. But high levels of chloride have been found to reduce the sensitivity of the starch-lacking stomata and isolated guard cell protoplasts (GCPs) from Allium to potassium ions, fusicoccin and abscisic acid. Furthermore, with high levels of chloride, malate anions disappear from the guard cells of Allium, a finding which contrasts with situation in Vicia where the stomatal sensitivity to K⁺ ions, fusicoccin and ABA is not influenced by Cl^- ions and malate levels are unaffected. It is suggested that the absence of malate as a proton yielding primer inhibits the mechanism of H⁺/K⁺ exchange in Allium.Abbreviations ABA abscisic acid - FC fusicoccin - GCPs guard cell protoplasts     

Gibberellic acid induces cytoplasmic acidification in maize coleoptiles

Indole-3-acetic acid (IAA), fusicoccin and weak acids all lower the cytoplasmic pH (pH_i) and induce elongation growth of maize (Zea mays L.) coleoptiles. Gibberellic acid (GA₃) also induces elongation growth and we have used confocal laser scanning microscopy to study the effects of GA₃ on pH_i employing the pH-indicator dyes, 2,7-bis(2-carboxyethyl)-5-(and-6) carboxyfluorescein and carboxy-semi-naphthorhodafluor-1. We confirm that GA₃ induces growth significantly in light-grown but only slightly or not at all in dark-grown coleoptiles. The growth induced by IAA treatment was similar in light- and dark-grown coleoptiles. The pH_i decreased by up to 0.6 units during the first 7 min of GA₃ or IAA treatment of both light- and dark-grown coleoptiles. Gibberellic acid inhibited IAA-induced growth of dark-grown coleoptiles. Hence, in dark-grown coleoptiles GA₃ may activate either directly or indirectly reactions that interfere with the signalling pathway leading to elongation growth. The possible role of pH_i in growth is discussed.Abbreviations ABA abscisic acid - AM acetoxymethyl ester - BCECF 2,7-bis(2-carboxyethyl)-5-(and-6) carboxyfluorescein - [Ca²⁺]_i cytoplasmic free calcium - GA_(n) gibberellin A_(n) - GA₃ gibberellic acid - IAA indole-3-acetic acid - PGR plant growth regulator - pH_i cytoplasmic pH - Pipes piperazine-N,N-bis[2-ethanesulfonic acid] - Snarf-1 carboxy-semi-naphthorhodafluor-1 We thank Dr R. King (CSIRO, Canberra) for providing the GA₁ and T. Phillips for processing the photographic material. H.R. Irving was supported by an Australian Research Council Research Fellowship and the work was supported by an Australian Research Council grant.     

Lack of correlation between overall protein synthesis and the onset of cell elongation in germinating embryos of Haplopappus gracilis

10⁻⁵M abscisic acid (ABA) completely inhibits germination or (if seeds deprived of integuments are used) embryo elongation in Haplopappus gracilis (Nutt.) Gray. Nevertheless, considerable rates of protein and RNA synthesis were found in embryos grown in abscisic acid, at least during the early hours after sowing. On the contrary, seeds grown in cycloheximide + fusicoccin (a powerful promoter of cell expansion), where protein synthesis is almost completely inhibited, show full protrusion of radicle, thus simulating a "germination" process. These results suggest that some of the most important events involved in seed germination, i.e. protein and RNA synthesis, and cell elongation which leads to radicle protrusion, may not necessarily be linked together and are possibly regulated by different control mechanisms. Moreover, when seeds or embryos are grown in abscisic acid + fusicoccin, protein synthesis is considerable, cell elongation is greater than in water controls at least for 12 h, and germination in its early stages appears to be normal; but DNA synthesis and cell division are not resumed, possibly since some other factor is required. All these findings propose a reevaluation of criteria for defining successful germination.     

Effect of auxin and abscisic acid on cell wall extensibility in maize coleoptiles

Plastic and elastic in-vitro extensibilities (E _pland E _el ) of cell walls from growing maize (Zea mays L.) coleoptile segments were measured by stretching frozen-thawed tissue, pre-extended to its in-vivo length, at constant force (creep test) in a custom-buildt extensiometer, equipped with a linear-displacement transducer. The indole-3-acetic acid (IAA)-induced change of E _pl (E _pl ) is strictly correlated with the growth rate for a period of 3–4 h. Subsequently, E _plremains constant while the growth rate is slowing down. Since this discrepancy can be accounted for by a growth-dependent reduction of osmotic pressure, it is concluded that E _plrepresents quantitatively the relative increase of in-vivo extensibility (cell wall loosening) involved in IAA-mediated cell growth over a much longer time. On the other side it is argued that the growth rate may not be strictly correlated with wall extensibility during long-term growth. Abscisic acid (ABA) inhibits segment growth induced by auxin, fusicoccin, or exogenous acid, and this effect can be quantitatively attributed to an ABA-mediated reduction of cell wall extensibility as determined by the E _plmeasurement. Both, IAA and ABA have no effect on total protein synthesis, RNA synthesis, and amount of osmotic solutes. Fusicoccin-induced proton excretion is only slightly inhibited by ABA. In contrast to ABA, growth inhibition by cycloheximide (CHI) is always much larger than the concomitant reduction of E _pl , indicating that a further growth parameter is also involved in the inhibition of cell growth by CHI. E _el is not affected by either IAA, ABA, or CHI. It is concluded that E _pl as determined by the applied method, represents a relative measure of the actual in-vivo extensibility of the growing cell wall at the very moment when the tissue is killed, rather than an average extensibility accumulated over some immediate-past period of time as suggested by Cleland (1984, Planta 160, 514–520). Hence, we further draw the conclusion that IAA and ABA control of cell growth can entirely be attributed to a modulation of cell wall extensibility by these hormones in maize coleoptiles.Abbreviations ABA ±abscisic acid - CHI cycloheximide - E _el , E_pl elastic and plastic in vitro extensibilities, respectively (E _el+E_pl=E_tot>) - FC fusicoccin - IAA indole-3-acetic acid     

Synthesis and metabolism of abscisic acid in detached leaves of Phaseolus vulgaris L. after loss and recovery of turgor

Metabolism of abscisic acid (ABA) was studied after wilting and upon recovery from water stress in individual, detached leaves of Phaseolus vulgaris L. (red kidney bean). Loss of turgor was correlated with accumulation of ABA and its metabolites, resulting in a 10-fold increase in the level of phaseic acid (PA) and a doubling of the level of conjugated ABA. The level of conjugated ABA in turgid leaves was no higher than that of the free acid. These results indicate that accumulation of ABA in wilted leaves resulted from a stimulation of ABA synthesis, rather than from a release from a conjugated form or from inhibition of the metabolism of ABA. The rate of synthesis of ABA was at its maximum between 2.5 and 5 h after turgor was lost, and slackened there-after. In wilted leaves, the rate of conversion of ABA to PA climbed steadly until it matched the rate of synthesis, after about 7.5 h. Upon rehydration of sections from wilted leaves, the rate of synthesis of ABA dropped close to zero within about 3 h, while the rate of conversion to PA accelerated. Formation of PA was two to four times faster than in sections maintained in the wilted condition; it reached a rate sufficient to convert almost one-half of the ABA present in the tissue to PA within 1 h. In contrast, the alternate route of metabolism of ABA, synthesis of conjugated ABA, was not stimulated by rehydration. The role of turgor in the stimulation of the conversion of ABA to PA was investigated. When leaves that had been wilted for 5 h were rehydrated to different degrees, the amount of ABA which disappeared, or that of PA which accumulated during the next 3 h, did not depend linearly on the water potential of the rehydrated leaf. Rather, re-establishment of the slightest positive turgor was sufficient to result in maximum stimulation of conversion of ABA to PA.Abbreviations ABA abscisic acid - DPA dihydrophaseic acid - PA phaseic acid - _leaf leaf water potential - osmotic pressure     

Radioimmunoassay for the determination of free and conjugated abscisic acid

The characterization and application of a radioimmunoassay specific for free and conjugated abscisic acid (ABA) is reported. The antibodies produced against a bovine serum albumin-(±)-ABA conjugate have a high affinity for ABA (K_a=1.3x10⁹l mol^-1). Trans, trans-ABA and related compounds, such as xanthoxin, phaseic acid, dihydrophaseic acid, vomifoliol or violaxanthin do not interfere with the assay. The detection limit of this method is 0.25x10^-12 mol ABA, the measuring range extends to 20x10^-12 mol, and average recoveries are 103%. Because of the high specificity of this immunoassay, no extract purification steps are required prior to analysis. Several hundred plants can be analyzed per day in a semi-automatic assay performance. ABA has been detected in all higher plant families examined, but was absent in the blue-green alga, Spirulina platensis, the liverwort Marchantia polymorpha, and two species of fungi.Abbreviations ABA abscisic acid - BHT 2.6-di-t-butyl-4-methyl phenol - TLC thin-layer chromatography - HSA human serum albumin Part 7 in the Series: Use of Immunoassay in Plant Science     

Ion channels in the membrane ofChara inflata

Summary Voltage-clamped steps in the electric potential difference (PD) across the membrane in cells of the green alga,Chara inflata, cause voltage- and time-dependent current flows, interpreted to arise from opening and closing of various types of ion channel in the membrane. With cells in the light, these channels are normally closed, and the resting PD is probably determined by the operation of an H⁺ efflux pump. Positive steps in PD from the resting level often caused the opening of K⁺ channels with sigmoid kinetics. The channels began to show opening when the PD–120 mV for an external concentration of K⁺ of 1.0mm. Return of the PD to the resting level caused closing of the channels with complex kinetics. Various treatments of the cell could cause these K⁺ channels to open, and remain open continuously, with the PD then lying closer to the Nernst PD for K⁺. The K⁺ channels have been identified by the blocking effects of TEA⁺. Another group of channels, probably Cl^– and Ca²⁺ associated with the action potential open when the PD is stepped to values less negative than –50 mV. Negative steps from the resting PD cause the slow opening, with a time course of seconds, of yet another type of channel, probably Cl^–.     

Gas chromatography-mass spectrometric determinations of abscisic acid levels in the cap and the apex of maize roots

Quantitative analyses of abscisic acid (ABA) in different parts of maize root tips (Zea mays L. cv. Kelvedon 33) were performed by mass fragmentography using the hexadeuterated analog of ABA as internal standard. It was found that the cap and the apex contained 36.1 g and 66.5 g ABA kg^–1 fresh weight, respectively. The possibility that the growth regulator formed in the cap and inhibiting the elongation of the extending zone of the root is ABA is discussed.Abbreviations ABA abscisic acid - ABA-D₆ hexadeuterated ABA - ABA-Me and ABA-D₆-Me methyl esters of ABA and ABA-D₆, respectively - GC-MS gas chromatograph(y)-mass spectrometry/spectrometer - IAA indol-3-yl-acetic acid - MF mass fragmentography - TMS trimethylsilyl     

Reversing the inhibitory effect of paclobutrazol on seed germination of Amaranthus paniculatus by GA3, ethephon or ACC

The germination of Amaranthus paniculatus seeds was inhibited by applying paclobutrazol, a specific inhibitor of gibberellin biosynthesis. This inhibition was markedly counteracted by gibberellin A₃ (GA₃), suggesting that endogenous gibberellins are required for germination in this species. The inhibitory effect of paclobutrazol was also overcome by ethephon (2-chloroethylphosphonic acid) or the precursor of ethylene biosynthesis, ACC (1-aminocyclopropane-l-carboxylic acid). Thus the physiological effect of gibberellin can be mimicked by ethylene released from ethephon or synthesised from exogenous ACC. It is suggested, that endogenous gibberellins are involved in germination of Amaranthus paniculatus seeds and that action of GA₃ can be substituted by ethylene.Abbreviations ACC 1-aminocyclopropane-l-carboxylic acid - AMO-1618 (2-isopropyl-5methyl-4-trimethylammoniumchloride)-phenyl-l-piperidinium-carboxylate - ancymidol -cyclopropyl--(4-methoxyphenyl)-5-pyrimidine methanol - chloromequat chloride (2-chloroethyl)trimethylammoniumchloride - ethephon 2-chloroethylphosphonic acid - GA gibberellin A₃ - paclobutrazol (2RS, 3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-lyl)pentan-3-ol - Phosphon D 2,4,dichlorobenzyl-tributhylphosphoniumchloride - tetcyclacis 5,(4-chlorophenyl)-3,4,5,9,10-pentaaza-tetracyclo)5,4,1,0,Z,6,0^8,11 dodeca-3,9-diene     

Effect of abscisic acid on membrane potential and transport of glucose and glycine in Lemna gibba G1

The membrane potential of Lemna gibba G1 was measured with a microelectrode; glucose and glycine uptake were measured with ¹⁴C-labeled substances. The membrane potential was increased by 85 mV on the average, after the plants had been pretreated with 10 M abscisic acid (ABA) for more than 30 min. This effect is not linked to the endogenous level of soluble sugars. The concentration of these soluble sugars was increased to more than 200% by pretreatment of the plants with ABA, however, the respiration of the plants was not affected. ABA stimulated uptake of glucose and glycine. Glucose- and glycine-dependent depolarization and repolarization of the membrane was altered: depolarization was less and repolarization was slower; during uptake of glycine, the first typical phase of repolarization was suppressed. The data suggest that ABA interferes with the primary steps of substrate uptake.Abbreviations ABA abscisic acid - FW fresh weight - IAA indole acetic acid - pd membrane potential difference - 1× perfusing solution (see methods) - H⁺ electrochemical proton gradient - pd solute-induced maximum depolarization of the membrane     

Acidification of the Medium Associated with Normal and Fusicoccin-Induced Seed Germination

Fusicoccin, a toxin stimulating cell enlargement and inducing proton extrusion in various plant tissues, has been shown to replace kinetin, gibberellic acid and red light in breaking seed dormancy. It also removes the inhibitory effect of abscisic acid. The present data also show that the stimulating effect of fucicoccin on embryo growth of decoated radish (Raphanus sativus L.) and maize (Zea mays) seeds and on the development of maize embryos is accompanied by an early, significant acidification of the medium. Acidification of the medium is also observed when fusicoccin reverses the abscisic acid-induced inhibition of germination. These results support the hypothesis that the mode of action of fusicoccin in promoting germination involves, as in stimulation of cell enlargement, the activation at the cell membrane level of proton extrusion processes. The physiological significance of fusicoccin-induced release of protons at the onset of germination is discussed in comparison with the results concerning the mechanism of action of fusicoccin on cell enlargement in other plant materials.     

Altered development of polysomal RNA activity in chick-pea (Cicer arietinum L.) embryonic axes. Effects of abscisic acid and temperature

The in vitro activity of polysomal polyadenylated RNA (poly(A)RNA) was studied using chick-pea (Cicer arietinum L.) embryonic axes subjected to treatments retarding germination (H₂O 30°C and abscisic acid [ABA] 30°C) or inducing a false germination (thiourea 30°C) in which normal protein synthesis and growth did not occur. All treatments induced a smaller proportion of poly(A)RNA compared with the control (H₂O 25°C). However, poly(A)RNA obtained in the presence of ABA had a similar in vitro activity to that of the control. The translation of mRNA from embryonic axes germinated at high temperatures was extensively blocked (70%) by methyl-7-guanosine-5-triphosphate, whereas mRNA translation from axes treated with H₂O-25°C and ABA was completely blocked (100%), indicating a greater cap dependence in the latter cases. Polyacrylamide gel electrophoresis showed that ABA and H₂O-30°C each induced the synthesis of a polypeptide with an approximate M_r of 32 kDa, probably a germination regulator. It is suggested that ABA and high temperatures could regulate germination at the translational level as well as affecting ionic-exchange properties, as has been previously demonstrated (Hernández-Nistal et al. 1983, Physiol. Plant. 57, 273–278).Abbreviations ABA abscisic acid - Poly (A) RNA polyadenylated RNA - TU thiourea     

The effects of abscisic acid on the maturation ofBrassica napus somatic embryos

Summary A comparison of embryos, cultured for increasing periods of time with and without abscisic acid (ABA), was undertaken to investigate, at the ultrastructural level, the influence of this growth regulator on the maturation of rapeseed (Brassica napus) somatic embryos. In the absence of ABA, the embryos germinated precociously while lipid bodies (LB), which were not numerous, soon degraded, as revealed by a depletion process associated with the appearance of morphologically mature glyoxysomes and an increase in the number of mitochondria. Moreover, a lack of protein bodies indicated that storage protein accumulation was not initiated under these conditions. On the contrary, the addition of ABA (10 M) induced marked modification of embryo metabolism. Indeed, ABA completely prevented precocious embryo germination and inhibited lipid reserve catabolism. Moreover, the formation of small vacuoles and proliferation of rough endoplasmic reticulum in their vicinity suggested the onset of storage protein accumulation. After 15 days in the presence of ABA, the embryos contained abundant lipid and protein bodies. Nevertheless, these somatic embryos were not exactly the same as their mature zygotic counterparts since differences were found in chloroplasts, amyloplasts, and nuclear structures. These observations suggest that additional factors might be required to obtain fully mature somatic embryos.Abbreviations ABA abscisic acid - ABM ABA medium - BM basal medium - LB lipid bodies - MS Murashige and Skoog (1962) - PB protein bodies - RER rough endoplasmic reticulum     

Mechanisms of fusicoccin action: A dominant role for secondary transport in a higher-plant cell

Fusicoccin (FC) is commonly thought to promote electrogenic H⁺ extrusion through its action on the H⁺-ATPase of the plant plasma membrane. Nonetheless, essential support from rigorous electrophysiological analysis has remained largely absent. The present investigation surveys the effects of FC on the charge transport properties at the membrane of a higher-plant cell — stomatal guard cells of Vicia faba L. — for which the electrical geometry is defined, and from which the voltage-dependent kinetic characteristic for the pump has been identified. Current-voltage (I-V) relations of the guard cells were determined before and during treatments with FC, and during brief exposures to NaCN plus salicylhydroxamic acid. Responses of the pump and of the ensemble of secondary transport processes were identified in the whole-membrane conductance-voltage relations and in the difference-current-voltage (dI-V) characteristic for the pump. In 0.1 mM K⁺, exposure to 10 M FC shifted guard-cell potentials negative by 29–61 mV. Current-and conductance-voltage profiles indicated limited changes in the pump I-V characteristic, an observation which was confirmed through explicit kinetic analysis of pump dI-V relations. However, the voltage response was accompanied by a 1.5-to 2.6-fold fall in membrane conductance. These results challenge conventional views of fusicoccin action by ascribing the electrical responses to reduced current passage through secondary transport pathways as well as to enhanced electrogenic ion pumping.Abbreviations and symbols Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - SHAM salicylhydroxamic acid - FC fusicoccin - V _m free-running membrane potential - G _m membrane slope conductance at V _m - (d)I-V (difference) current-voltage (relation) - G-V slope conductance-voltage (relation)