Cytoplasmic Ca2+, K+, Cl-, and NO3- Activities in the Liverwort Conocephalum conicum L. at Rest and during Action Potentials

Intracellular Ca2+, K+, Cl-, and NO3- activities were measured with ion-selective microelectrodes in the liverwort Conocephalum conicum L. at rest, during dark/light changes, and in the course of action potentials triggered by light or electrical stimuli. The average free cytosolic Ca2+ concentration was 231 [plus or minus] 65 nM. We did not observe any light-dependent changes of the free cytosolic Ca2+ concentration as long as no action potential was triggered. During action potentials, on average a 2-fold increase of the free cytoplasmic Ca2+ concentration was recorded. Intracellular K+ activity was 76 [plus or minus] 10 mM. It did not depend on K+ concentration changes in the bath solution between 0.1 and 10 mM. The average equilibrium potential for K+ in the standard medium containing 1 mM K+ was -110 mV, which differed significantly from the resting potential of -151 [plus or minus] 2 mV. During action potentials, either a slight decrease or no changes in intracellular K+ activity were recorded. The average Cl- activity was 7.4 [plus or minus] 0.2 mM in the cytoplasm and 43.5 [plus or minus] 7 mM in the vacuole. The activities of NO3- were 0.63 [plus or minus] 0.05 mM in the cytoplasm and 3.0 [plus or minus] 0.3 mM in the vacuole. For both anions the vacuolar activity was 5 to 6 times higher than the cytoplasmic activity. After the light was switched off both the Cl- and the NO3- activity showed either no change or a slight increase. Illumination caused a gradual return to previous values or no change. During action potentials a slight decrease of intracellular Cl- activity was recorded. It was concluded that in Conocephalum, as in characean cells, chloride channels are involved in the depolarization phase of the action potentials. We discuss a model for the ion fluxes during an action potential in Conocephalum.     

Two distinct signaling pathways participate in auxin-induced swelling of pea epidermal protoplasts

Protoplast swelling was used to investigate auxin signaling in the growth-limiting stem epidermis. The protoplasts of epidermal cells were isolated from elongating internodes of pea (Pisum sativum). These protoplasts swelled in response to auxin, providing the clearest evidence that the epidermis can directly perceive auxin. The swelling response to the natural auxin IAA showed a biphasic dose response curve but that to the synthetic auxin 1-naphthalene acetic acid (NAA) showed a simple bell-shaped dose response curve. The responses to IAA and NAA were further analyzed using antibodies raised against ABP1 (auxin-binding protein 1), and their dependency on extracellular ions was investigated. Two signaling pathways were resolved for IAA, an ABP1-dependent pathway and an ABP1-independent pathway that is much more sensitive to IAA than the former. The response by the ABP1 pathway was eliminated by anti-ABP1 antibodies, had a higher sensitivity to NAA, and did not depend on extracellular Ca(2+). In contrast, the response by the non-ABP1 pathway was not affected by anti-ABP1 antibodies, had no sensitivity to NAA, and depended on extracellular Ca(2+). The swelling by either pathway required extracellular K(+) and Cl(-). The auxin-induced growth of pea internode segments showed similar response patterns, including the occurrence of two peaks in the dose response curve for IAA and the difference in Ca(2+) requirements. It is suggested that two signaling pathways participate in auxin-induced internode growth and that the non-ABP1 pathway is more likely to be involved in the control of growth by constitutive concentrations of endogenous auxin.     

Conjugation of Indole-3-Acetic Acid (IAA) in Wild-Type and IAA-Overprodcing Transgenic Tobacco Plants,and Identification of the Main Conjugates by Frit-Fast Atom Bombardment Liquid Chromatography-Mass Spectrometry

Transgenic plants overproducing indole-3-acetic acid (IAA) from expression of the Agrobacterium tumefaciens T-DNA IAA biosynthesis genes were used to study the conjugation of IAA. At the 11-node stage, free IAA, as well as ester- and amide-conjugated IAA, was analyzed in wild-type tobacco SR1 and in transgenic plants denoted 35S-iaaM/iaaH (line C) and 35S-iaaM x 35S-iaaH (line X). The transgenic plants contained increased levels of both free and conjugated IAA, and the main increase in IAA conjugates occurred in amide conjugates. Two amide conjugates were identified by fritfast atom bombardment liquid chromatography-mass spectrometry as indole-3-acetylaspartic acid (IAAsp) and indole-3-acetylglutamic acid (IAGlu), and one ester conjugate was identified as indole-3-acetylglucose. IAAsp and IAGlu were also identified as endogenous substances in wild-type plants. In wild-type plants, the percent of total IAA in the free form was significantly higher in young leaves (73 [plus or minus] 7%, SD) than in old leaves (36 [plus or minus] 8%), whereas there was no difference between young (73 [plus or minus] 8%) and old internodes (70 [plus or minus] 9%). In IAA-overproducing transformants, both free and conjugated IAA levels were increased, but the percent free IAA was maintained constant (57 [plus or minus] 10%) for both leaves and internodes, independent of the total IAA level or tissue age. These results suggest that synthesis or transport of IAA conjugates is regulated in the vegetative wild-type plant, and that different organs possess a unique balance between free and conjugated IAA. The IAA-overproducing plant, however, acquires a lower proportion of free IAA in the stem and younger leaves, presumably determined by a higher conjugation in those tissues compared with wild type.     

Simultaneous Measurement of Intracellular pH and K+ or NO3- in Barley Root Cells Using Triple-Barreled,Ion-Selective Microelectrodes

The manufacture and use of triple-barreled microelectrodes, which are capable of simultaneous in vivo measurement of intracellular pH and the activities of K+ or NO3- and cell membrane potential (Em), are described. Scanning electron micrographs showed that the three tips were aligned and that the overall tip diameter was approximately 0.8 [mu]m. When filled with 100 mM KCl, all three barrels simultaneously reported identical transmembrane potentials, showing that all three tips were located in the same subcellular compartment. Intracellular estimates of Em in barley (Hordeum vulgare L. cv Klaxon) root epidermal cells obtained with these triple-barreled microelectrodes were indistinguishable from those obtained using single- or double-barreled microelectrodes. Measurements made with triple-barreled K+ and pH-selective microelectrodes in root cells of 7-d-old barley plants grown in a nutrient solution containing 0.5 mM K+ yielded cytosolic and vacuolar populations having mean K+ activity values of 71.3 and 68.7 mM, respectively. The associated mean pH values ([plus or minus]SE) were 7.26 [plus or minus] 0.06 (cytosol) and 5.18 [plus or minus] 0.08 (vacuole). Analysis of whole-tissue digests confirmed the microelectrode measurements. Measurements made using triple-barreled pH- and nitrate-selective microelectrodes confirmed earlier double-barreled measurements of pH and nitrate in barley root epidermal cells growing in 10 mM nitrate.     

钙在IAA诱导绿豆下胚轴原生质体膨大过程中的作用

含钙培养液(对照)和仅用IAA处理的原生质体的体积和~(45)Ca~(2 )放射性强度均无变化。IAA处理含钙培养液中的原生质体,5min后~(45)Ca~(2 )积累明显增多,体积开始膨大。处理30min时~(45)Ca~(2 )积累最多,此时原生质体的膨大效应最好;随后~(45)Ca~(2 )积累和膨大效应逐渐下降。K~ 、Zn~(2 )、Ba~(2 )、Mg~(2 )等也可在一定程度上代替Ca~(2 )使原生质体体积膨大。原生质体的吸水在膨大中起着一定作用。EGTA、LaCl_3和verapamil均抑制IAA诱导的原生质体~(45)Ca~(2 )积累和体积膨大。说明Ca~(2 )可能在6-BA诱导原生质体膨大的过程中起着重要作用。     

Phosphorylation of Na,K-ATPase by acetyl phosphate and inorganic phosphate. Sidedness of Na+, K+ and nucleotide interactions and related enzyme conformations.

The effects of K+, Na+ and nucleotides (ATP or ADP) on the steady-state phosphorylation from [32P]Pi (0.5 and 1 mM) and acetyl [32P]phosphate (AcP) (5 mM) were studied in membrane fragments and in proteoliposomes with partially purified pig kidney Na,K-ATPase incorporated. The experiments were carried out at 20 degrees C and pH 7.0. In broken membranes, the Pi-induced phosphoenzyme levels were reduced to 40% by 10 mM K+ and to 20% by 10 mM K+ plus 1 mM ADP (or ATP); in the presence of 50 mM Na+, no E-P formation was detected. On the other hand, with AcP, the E-P formation was reduced by 10 mM K+ but was 30% increased by 50 mM Na+. In proteoliposomes E-P formation from Pi was (i) not influenced by 5-10 mM K+cyt or 100 mM Na+ext, (ii) about 50% reduced by 5, 10 or 100 mM K+ext and (iii) completely prevented by 50 mM Na+cyt. Enzyme phosphorylation from AcP was 30% increased by 10 mM K+cyt or 50 mM Na+cyt; these E-P were 50% reduced by 10-100 mM K+ext. However, E-P formed from AcP without K+cyt or Na+cyt was not affected by extracellular K+. Fluorescence changes of fluorescein isothiocyanate labelled membrane fragments, indicated that E-P from AcP corresponded to an E2 state in the presence of 10 mM Na+ or 2 mM K+ but to an E1 state in the absence of both cations. With pNPP, the data indicated an E1 state in the absence of Na+ and K+ and also in the presence of 20 mM Na+, and an E2 form in the presence of 5 mM K+. These results suggest that, although with some similarities, the reversible Pi phosphorylation and the phosphatase activity of the Na,K-ATPase do not share the whole reaction pathway.     

Epidermal Cells Do Not Contribute to Auxin-Induced Ethylene Production in Mung Bean Stem Sections

Auxin-induced and 1-aminocyclopropane-1-carboxylic acid (ACC)-dependentethylene production in mung bean (Vigna radiata [L] Wilczek)hypocotyl sections, from which epidermis had been removed, wasinvestigated. Ethylene production in hypocotyl sections withoutepidermis was induced by treatment with IAA, and also occurredfrom exogenously supplied ACC in the presence of 0.2 M mannitol.Isolated epidermal strips alone failed to produce substantialamounts of ethylene in response to IAA or from exogenous ACC.3,4-[¹⁴C]-Methionone was incorporated into both ACC and ethylenein peeled sections treated with IAA, but not in the isolatedepidermal strips. Radioactive ACC, however, was detected inthe epidermal strips separated from the unpeeled sections previouslyfed with 3,4-[¹⁴C]-methionine in the presence of IAA. We concludethat the Site of auxin-induced ethylene production is not inthe epidermis, but in other hypocotyl cells, and that epidermalcells lack the activity which converts ACC to ethylene. (Received January 28, 1985; Accepted May 4, 1985)     

Sulphate transport by H+ symport and by the dicarboxylate carrier in mitochondria.

1. Swelling of mitochondria was induced in non-respiring mitochondria by 30 mM or more Na2SO4 or by respiration in the presence of K2SO4. Respiration-drive swelling resulted in loss of respiratory control. Sulphate, when present at 10 mM concentration, promoted the release of accumulated Ca2+. 2. Swelling was prevented by N-ethylmaleimide and formaldehyde, known inhibitors of the phosphate carrier. Sulphate-induced swelling was more sensitive to the inhibitors than was phosphate-induced swelling. At lower concentration of sulphate, 5 mM, an alkalinisation of the medium was observed in addition of sulphate, indicating H+-sulphate symport. There was competition between sulphate and phosphate for transport by this mechanism. It is suggested that sulphate may be transported, though at a comparatively slow rate, by the phosphate carrier. 3. Uptake of sulphate was stimulated when preceded by energy-dependent accumulation of Ba2+, especially when acetate was also present, indicating precipitation of BaSO4 in the matrix. Using this system the influx of sulphate was studied at lower concentrations, 10 mM or less. the contributions of the H+ symporter (sensitive to N-ethylmaleimide) and the dicarboxylate carrier (sensitive to butylmalonate) could then be studied. The dicarboxylate carrier had a lower Km and was mainly responsible for sulphate transport at lower concentration range. At 10 mM-sulphate the transport rates by the two systems appeared to be similar; at still higher concentrations the H+ symporter may become more important.     

钙在IAA诱导绿豆下胚轴原生质体膨大过程中的作用

This paper studied on the role of calcium in IAA-induced swelling of protoplasts isolated from hypocotyl in etiolated mung bean (Phaseolus radiatus L.) seedlings. Protoplasts incubated in CaCl2-bearing medium without hormone maintained a constant volume and a consistent intensity of 45Ca2+ radioactivity. To treat with IAA, they began to swell and continually swelled to the maximum volume 30 minutes later (Fig. 2). However, the protoplasts could not swell when IAA was added into the medium without CaCl2 (Fig. 1). It was suggested that Ca2+ may be necessary for IAA to induce protoplast swelling. And also, IAA enabled the protoplasts to swell in less extent with K+, Zn2+, Ba2+ or Mg2+ instead of Ca2+ (Fig. 3). Radioisotope experiments showed that K+ influx increased when K+ replaced Ca2+ (Fig. 4), and water absorption plays a role in the swelling (Fig. 5). 45Ca2+ accumulation in protoplasts treated by IAA was much higher than that of control, and the time course of 45Ca2+ accumulation was similar to that of protoplasts swelling (Fig. 6). 45Ca2+ level and the swelling of protoplasts sharply declined when EGTA, verapamil or LaCl3 was added into the medium (Table 1, 2 and 3). These results indicated that Ca2+ may play an important role in IAA-induced swelling.     

A Comparison of Freezing Injury in Oat and Rye: Two Cereals at the Extremes of Freezing Tolerance

A detailed analysis of cold acclimation of a winter rye (Secale cereale L. cv Puma), a winter oat (Avena sativa L. cv Kanota), and a spring oat cultivar (Ogle) revealed that freezing injury of leaves of nonacclimated seedlings occurred at -2[deg]C in both the winter and spring cultivars of oat but did not occur in winter rye leaves until after freezing at -4[deg]C. The maximum freezing tolerance was attained in all cultivars after 4 weeks of cold acclimation, and the temperature at which 50% electrolyte leakage occurred decreased to -8[deg]C for spring oat, -10[deg]C for winter oat, and -21[deg]C for winter rye. In protoplasts isolated from leaves of nonacclimated spring oat, expansion-induced lysis was the predominant form of injury over the range of -2 to -4[deg]C. At temperatures lower than -4[deg]C, loss of osmotic responsiveness, which was associated with the formation of the hexagonal II phase in the plasma membrane and subtending lamellae, was the predominant form of injury. In protoplasts isolated from leaves of cold-acclimated oat, loss of osmotic responsiveness was the predominant form of injury at all injurious temperatures; however, the hexagonal II phase was not observed. Rather, injury was associated with the occurrence of localized deviations of the plasma membrane fracture plane to closely appressed lamellae, which we refer to as the "fracture-jump lesion." Although the freeze-induced lesions in the plasma membrane of protoplasts of spring oat were identical with those reported previously for protoplasts of winter rye, they occurred at significantly higher temperatures that correspond to the lethal freezing temperature.     

Insulin release and eflux of [32P]Phosphate from islets of rat Langerhans treated with iodoacetic acid and the anomers of D-glucose.

To clarify the insulin-releasing mechanism, we studied insulin release and the efflux of [32P]phosphate by glucose at 0.1 mM/min of gradient level or at 16.7 mM, and other metabolism in islets of rat Langerhans. When treated with 1 mM iodoacetic acid (IAA) plus the anomers of D-glucose at 2.8 mM for 6 min at 37 degrees C, islets elicited insulin at half the control rate under the step-wise stimulation by glucose and at the same rate as the control under the slow-rise stimulation by glucose. Using islets treated with IAA plus the alpha anomer at 16.7 mM, the step-wise stimulation secreted insulin at half a rate of the control and the slow-rise stimulation at the rate lower than the control, which was not significantly different from the control rate. Treatment with IAA plus the beta anomer at 16.7 mM inhibited insulin release under both types of stimulations by glucose. The step-wise stimulation caused the same rapid efflux of [32P]phosphate from IAA-treated islets as from the control islets, except for islets treated with IAA plus the beta anomer at 16.7 mM. The rate of glucose utilization in islets was inhibited by all IAA-treatments to the same extent, being merely half the control rate. Treatments with IAA plus the anomers at 16.7 mM significantly reduced the formation of [3H]-cAMP and the activity of protein phosphokinase in islets, while in the presence of the anomers at 2.8 mM IAA produced no significant effect. Neither IAA-treatments altered the uptake of 45Ca and the ATP content in islets. The uptake of [14C]IAA was significantly enhanced by the presence of the beta anomer at 16.7 mM to two times the control level. On the basis of these results, we suggested that the B cell might contain both glucoreceptors and rate-sensors of glucose controlling insulin release and the former might be less sensitive to IAA as compared with the latter.     

Some properties of the H-ATPase activity present in root plasmalemma of Avena sativa L. Two different enzymes or one enzyme with two ATP sites?

The effects of Mg2+, K+ and ATP on a H-ATPase activity from a native plasmalemma fraction of oat roots were explored at 20 degrees C and pH 6.5. In the presence of 3 mM ATP and no K+, H-ATPase activity vs. [Mg2+] approached a monotonic activation but it became biphasic, with a decline above 3 mM Mg2+, in the presence of 20 mM K+. Mg2+ inhibition occurred also in K-free solutions when [ATP] was lowered to 0.05 mM. Also, an apparent monotonic H-ATPase activation by [K+] at 3.0 mM ATP was transformed in biphasic (inhibition by high [K+]) when [ATP] was reduced to 0.05 mM. The best fits of the ATP stimulation curves of hydrolysis satisfied the sum of two Michaelian functions where that with higher affinity had lower Vmx. Taking into consideration all conditions of activity assay, the high-affinity component (1) had a Km about 11-16 microM and a Vmx around 0.14-0.28 mumol Pi/mg per min whereas that with lower affinity (2) had a Km of 220-540 microM and a Vmx of 0.5-1.0 mumol Pi/mg per min. Km2 was markedly affected by the [K+] and [Mg2+]; at optimal concentrations of these cations (1 mM Mg2+ and 10 mM K+) it had a value of 235 +/- 24 microM which was increased to 540 +/- 35 microM at 20 mM [Mg2+] and 60 mM [K+]. In addition, Vmx1 was reduced to about a half when the concentrations of Mg2+ and K+ were increased to inhibitory levels. These results could be explained by the existence of two different enzymes or one enzyme with two ATP sites. In the second case, we could not tell at this stage if both are catalytic or one is regulatory.     

Ca2+-Calmodulin Modulates Ion Channel Activity in Storage Protein Vacuoles of Barley Aleurone Cells

Many plant ion channels have been identified, but little is known about how these transporters are regulated. We have investigated the regulation of a slow vacuolar (SV) ion channel in the tonoplast of barley aleurone storage protein vacuoles (SPV) using the patch-clamp technique. SPV were isolated from barley aleurone protoplasts incubated with CaCl2 in the presence or absence of gibberellic acid (GA) or abscisic acid (ABA). A slowly activating, voltage-dependent ion channel was identified in the SPV membrane. Mean channel conductance was 26 pS when 100 mM KCl was on both sides of the membrane, and reversal potential measurements indicated that most of the current was carried by K+. Treatment of protoplasts with GA3 increased whole-vacuole current density compared to SPV isolated from ABA- or CaCl2-treated cells. The opening of the SV channel was sensitive to cytosolic free Ca2+ concentration ([Ca2+]i) between 600 nM and 100 [mu]M, with higher [Ca2+]i resulting in a greater probability of channel opening. SV channel activity was reduced greater than 90% by the calmodulin (CaM) inhibitors W7 and trifluoperazine, suggesting that Ca2+ activates endogenous CaM tightly associated with the membrane. Exogenous CaM partially reversed the inhibitory effects of W7 on SV channel opening. CaM also sensitized the SV channel to Ca2+. In the presence of ~3.5 [mu]M CaM, specific current increased by approximately threefold at 2.5 [mu]M Ca2+ and by more than 13-fold at 10 [mu]M Ca2+. Since [Ca2+]i and the level of CaM increase in barley aleurone cells following exposure to GA, we suggest that Ca2+ and CaM act as signal transduction elements mediating hormone-induced changes in ion channel activity.     

Enhancement by Potassium of Carbachol-Stimulated Inositol Phospholipid Breakdown in Rat Cerebral Cortical Miniprisms: Comparison with Other Depolarising Agents

Increasing the [K+] in the assay medium from 5.7 to 17.8 mM produces a large enhancement of the inositol phospholipid breakdown response to the muscarinic agonist carbachol in rat cerebral cortical miniprisms, with minor effects on basal inositol phospholipid breakdown. This effect is also found with Rb+. The enhancement by a raised [K+] is not accompanied by a change in the composition of the labelled polyphosphoinositides. The carbachol-stimulated inositol phospholipid breakdown at 17.8 and 42.7 mM K+ was antagonised by veratrine (5-80 microM), 4-aminopyridine (5 mM), and tetraethylammonium (20 mM). These compounds, however, also inhibited the binding of [3H]quinuclidinyl benzilate to cortical membranes. BRL 34915 (0.2-20 microM) was without significant effect on carbachol-stimulated inositol phospholipid breakdown at either 5.7 or 17.8 mM K+.Mg2+ (10 mM) considerably reduced the carbachol-stimulated inositol phospholipid breakdown at 17.8, but not 42.7, mM K+. Inositol phospholipid breakdown was also stimulated, albeit to a small extent, by L-glutamate (100-3,000 microM) and quisqualate (1-100 microM), with the stimulation being additive to that produced by carbachol at both 5.7 and 17.8 mM K+. N-Methyl-D-aspartate (10-1,000 microM in Mg2+-free medium) had no significant effect on basal inositol phospholipid breakdown and had little or no effect on carbachol-stimulated inositol phospholipid breakdown at either 5.7 or 17.8 mM K+. It is concluded that it may not be correct to ascribe wholly the enhancement by K+ of carbachol-stimulated inositol phospholipid breakdown to the tissue-depolarising actions of this ion and that other actions of K+ may be involved.     

Membrane-Delimited Phosphorylation Enables the Activation of the Outward-Rectifying K Channels in Motor Cell Protoplasts of Samanea saman

Outward-rectifying K channels activated by membrane depolarization (Kout or KD channels) control K+ efflux from plant cells. To find out to what extent phosphorylation is required for the activity of these channels, the patch-clamp method was applied to protoplasts from the legume Samanea saman in both whole-cell and isolated-patch configurations. In the absence of either Mg2+ or ATP in the "cytosolic" solution, the KD channel activity declined completely within 15 min. This decline could be reversed in excised, inside-out patches by restoring MgATP (1 mM) to the cytoplasmic side of the membrane. Mg2+ (1 mM) plus 5[prime]-adenylylimidodiphosphate (1 mM), a nonhydrolyzable ATP analog, did not substitute for ATP. Mg2+ (1 mM) plus adenosine 5[prime]-O-(3-thiotriphosphate) (25 to <100 [mu]M), an irreversibly thiophosphorylating ATP analog, sustained channel activity irreversibly. 1-(5-IsoquinolinesulphonyI)-2- methylpiperazine (100 [mu]M), a broad-range kinase inhibitor, blocked the activity of KD channels in the presence of MgATP. These results strongly suggest that the activation of the outward-rectifying K channels by depolarization depends critically on phosphorylation by a kinase tightly associated with the KD channel.     

A chlorate-hypersensitive, high nitrate/chlorate uptake mutant of Arabidopsis thaliana

N-ethylmaleimide (NEM) Lit 10-100 μ M led to a strong inhibition of the auxin-induced elongation growth of colcoptile segments, while fusicoccin-enhanced growth was not affected. Growth inhibition occurred only if NEM and auxin were allowed to act simultaneously. Preincubation of plant segments with NEM in the absence of auxin caused no inhibition of a subsequent growth stimulation by auxin, whenever NEM was removed before the application of IAA. However, preincubation with NEM plus auxin led to a remaining growth inhibition, which could not be reversed by a second auxin incubation in the absence of NEM. Fusicoccin added to NEM- plus auxin-treated segments was able to restore growth. It is suggested that auxin causes the unmasking of essential SH-groups of a protein to which NEM links covalently. thus inhibiting the growth process. This assumption was further supported by labeling experiments wish [¹⁴C]-NEM using membranes of maize ( Zea mays L. cv. Inraplus) coleoptiles. Two membrane fractions (S₂= 480-1900 g; S₄= 4300-15000 g) revealed a significantly higher [¹⁴C]-NEM labeling in the presence of auxin (2,4-diehlorophe-noxyacctic acid compared to 2,6 dichlorophenoxyacetic acid). This effect disappeared when the membranes were previously washed with EGTA [ethyleneglycolbis-(β-aminoethylether)-N,N,Nr',N'-tetraacetic acid]. The auxin-induced sensitization of coleoptilc segments against thiol-reagents and the auxin-induced expression of SH-groups of proteins of isolated membranes from coleoptiles arc suggested to be events involved in the primary action of auxins.     

Fusicoccin- and IAA-induced elongation growth share the same pattern of K+ dependence

The dependence of growth induced by the fungal toxin fusicoccin (FC) on the K+ content of the incubation medium was investigated in abraded maize coleoptiles. If the divalent ion Ca2+ was included in the bathing medium, no FC-induced growth occurred in the absence of K+, whereas a strong response was detected in presence of K+. The optimal K+ concentration was in the range of 1-10 mM. With the exception of Rb+, none of the other alkali ions (Na+, Li+, Cs+) could replace for K+ in sustaining FC-induced growth. The potassium channel blocker tetraethylammonium (TEA) reversibly inhibited FC-induced growth. As shown earlier for auxin-induced growth, no strict potassium dependence of FC-triggered elongation was observed in Ca2+ -free media. However, TEA abolished this apparently K+ independent FC-induced growth. It is concluded that FC-induced growth, like auxin-induced growth, requires K+ uptake through K+ channels.     

New methods to analyse auxin-induced growth II: The swelling reaction of protoplasts – a model system for the analysis of auxin signal transduction?

A method for monitoring the time course of auxin-induced volume changesby protoplasts at a high temporal resolution was developed for Zeamays coleoptile protoplasts. Auxins, like indole-3-acetic acid(IAA), induce a rapid change in volume. Immediately after addition ofthis auxin, a transient shrinkage was observed, followed by a long-termswelling response. This reaction occurred in the same time window as thetypical auxin growth response of intact coleoptiles. Active auxins, like1-naphthalene acetic acid (1-NAA) and 4-chloroindole-3-acetic acid(4-Cl-IAA), caused similar volume changes, whereas the inactive analogue2-naphthalene acetic acid (2-NAA) had no effect. The phytotoxinfusicoccin (FC) induced a rapid swelling response. We conclude that thissingle cell system is very adequate to analyse mechanisms of auxinsignal transduction.     

Microautoradiographic localisation of [3H]sucrose and [3H]mannitol in Robinia pseudoacacia pulvinar tissues during phytochrome-mediated nyctinastic closure

Summary. We have analysed the incorporation of [³H]sucrose and [³H]mannitol in pulvinar motor cells of Robinia pseudoacacia L. during phytochrome-mediated nyctinastic closure. Pairs of leaflets, excised 2 h after the beginning of the photoperiod, were fed with 50 mM [³H]sucrose or [³H]mannitol, irradiated with red (15 min) or far-red (5 min) light and placed in the dark for 2–3 h. Label uptake was measured in whole pulvini by liquid scintillation counting. The distribution of labelling in pulvinar sections was assessed by both light and electron microautoradiography. [³H]Sucrose uptake was twice that of [³H]mannitol incorporation in both red- and far-red-irradiated pulvini. In the autoradiographs, [³H]sucrose and [³H]mannitol labelling was localised in the area from the vascular bundle to the epidermis, mainly in vacuoles, cytoplasm, and cell walls. Extensor and flexor protoplasts displayed a different distribution of [³H]sucrose after red and far-red irradiation. Far-red light drastically reduced the [³H]sucrose incorporation in extensor protoplasts and caused a slight increase in internal flexor protoplasts. After red light treatment, no differences in [³H]sucrose labelling were found between extensor and flexor protoplasts. Our results indicate a phytochrome control of sucrose distribution in cortical motor cells and seem to rule out the possibility of sucrose acting as an osmoticum. Correspondence and reprints: Unidad de Fisiología Vegetal, Facultad de Biología, Universidad de Barcelona, Avenida Diagonal 645, 08028 Barcelona, Spain.     

Oxidative Stress Affects [alpha]- Tocopherol Content in Soybean Embryonic Axes upon Imbibition and following Germination

The content of [alpha]-tocopherol ([alpha]T) in isolated soybean (Glycine max, var Hood) embryonic axes was measured upon germination. Dry, high-vigor axes contained 1.2 [plus or minus] 0.1, nmol/axis and after an increase during the initial 6 h of imbibition, there was a decline to 1.0 [plus or minus] 0.1 nmol/axis at 24 h of incubation. Incubation in the presence of the redox-cycling agent paraquat (4 mM) for 24 h increased the [alpha]T content to 1.9 [plus or minus] 0.2 nmol/axis. When the incubation medium was supplemented with 500 [mu]M Fe-EDTA over 24 h, the content of [alpha]T increased to 1.8 [plus or minus] 0.1 nmol/axis. Isolated axes from soybean seeds stored for 56 months contained 6.5 [plus or minus] 0.3 nmol of [alpha]T/axis after 24 h of imbibition as compared to 1.0 [plus or minus] 0.1 nmol of [alpha]T/axis in axes from soybean seeds stored for 8 months. In all of these experimental situations, oxidant production as assessed in vivo by a fluorometric assay was increased by 4 mM paraquat (8-fold), 500 [mu]M iron (2-fold), and 56 months of storage (4-fold) after 24 h of imbibition. The data presented here suggest that the cellular content of [alpha]T is physiologically adjusted as a response to conditions of oxidative stress.