Effects of Osmotic Stress, Salt Stress and IAA on the Regeneration of the Resting Membrane Potential after Excision of a Segment from an Intact Plant

Effects of osmotic stress, salt stress and IAA on the regenerationprocess of the transmembrane potential across the xylem/symplastinterface (V_px) of newly excised hypocotyl segments of Vignaseedlings were examined by means of the xylem perfusion method.It took about 8 h under ordinary conditions for an excised segmentto regenerate a membrane potential comparable with that of anintact seedling. Osmotic stress imposed by perfusion of 100-200mM sorbitol solution seemed to accelerate this process. Thiseffect diminished with the removal of sorbitol from the perfusionsolution. The increase in the negativity of V_px in this regenerationprocess resulted from the increases in both passive (V_px inN₂) and respiration-dependent components (V_px). NaCl (50–100mM) did not accelerate the regeneration of the total membranepotential, but significantly promoted an increase in V_px, i.e.electrogenic xylem pump activity. Perfusion of KC1 (50–100mM) or IAA (10^–4M) shortened the regeneration phase upto 2–3 h. The increase in total V_px under salt stressor IAA mainly resulted from the increase of V_px. The effectof those agents on the V_px is discussed, as is the questionof whether the increase in PD after excision should be interpretedas a recovery or regeneration from injury by excision, or asa so-called release from hormonal control. (Received September 3, 1987; Accepted March 14, 1988)     

Effects of Exogenous Auxin on the Regulation of Elongation Growth of Excised Segments of Vigna Hypocotyls under Osmotic Stress

IAA applied simultaneously with osmotica greatly enhanced theadaptive recovery of the elongation growth of segments of Vignahypocotyls during osmotic stress irrespective of whether ornot absorbable solutes were present. IAA stimulated both thesurface pump and the xylem pump, which have been shown to bestimulated by osmotic stress and to control the yielding ofthe cell wall and the absorption of solutes. Thus, wall extensibilityand the effective turgor were further enhanced under osmoticstress in the presence of IAA. These results indicate that thesimultaneous presence of IAA can reduce the inhibition of growthby osmotic stress, and they support numerical predictions basedon the apoplast canal model. The mechanism involved in the rapidrecovery of growth is discussed. ¹ Present address: Research Centre, Guangxi Agricultural University,Xiu Ling Rd., Nanning, Guangxi 530005 China. ² Present address: Biology Institute, Department of GeneralEducation, Nagoya University, 1 Furo-cho, Chikusa-ku, Nagoya,464 Japan. ³ Present address: Graduate School of Integrated Science, YokohamaCity University, 22-2, Seto, Kanazawa-ku, Yokohama, 236 Japan.     

Regulation of Elongation Growth of Excised segments of Vigna Hypocotyl under Osmotic Stress in the Absence or Presence of Absorbable Solute

The regulation of elongation growth in excised segments of Vignahypocotyl under osmotic stress was investigated by means ofthe xylem perfusion and pressure-jump method. When subjectedto osmotic stress in the absence of absorbable solutes (100mM sorbitol only) or in the presence of absorbable solutes (70mM sorbitol plus 30 mM sucrose, or 70 mM sorbitol plus 15 mMKC1), the hypocotyl segments immediately began to shrink. Thehyper-polarizations of the transmembrane potentials (V_px andV_ps) took place at once. Within 40–60 minutes, the segmentsresumed growth. In the presence of absorbable solute, therewas an obvious increase in the effective turgor (Pⁱ–Y'),but the physiological wall extensibility () increased only slightly.Conversely, in the absence of absorbable solute, increasedsignificantly but (Pⁱ–Y') decreased. The results suggestthat the recovery of growth of an excised segment under osmoticstress is mainly due to the change in in the absence of absorbablesolute, and to the change in (P¹–Y') in the presence ofabsorbable solute, and that the two respiration-dependent protonpumps play important roles in these recovery processes. (Received April 28, 1989; Accepted August 24, 1989)     

NADP+-Dependent Sorbitol Dehydrogenase Found in Apple Leaves

An NADP⁺-dependent sorbitol dehydrogenase that catalyzes sorbitoland glucose was found in apple leaves. The partially purifiedenzyme had optimum activity at pH 9.6 and a K_m value of 128mM for sorbitol. Among the polyols studied, this enzyme showedthe most activity for sorbitol. ¹This paper is contribution A-173 of the Fruit Tree ResearchStation. (Received June 4, 1984; Accepted July 31, 1984)     

Characterization of Hexose Transporter for Facilitated Diffusion of the Tonoplast Vesicles from Pear Fruit

Tonoplast vesicles were prepared from the flesh tissue of maturepear fruit. Sugar uptakes into the vesicles determined by twodifferent methods, the membrane and the gel filtration methods,were quite similar. The uptake was highest for glucose and subsequently,in order, for fructose, sucrose and sorbitol. It was not stimulatedby addition of ATP, although the vesicles could create a protongradient. However, the uptakes were significantly inhibitedby p-chloromercuribenzene sulphonate (PCMBS, SH-reagent andinhibitor of sugar transporter). Further, the PCMBS-sensitiveuptakes of glucose and fructose saturated with their increasedconcentrations. Thus, these PCMBS-sensitive uptakes are mediatedby the transporter of facilitated diffusion. The uptakes ofglucose or fructose each had two K_m values. K_m values for glucosewere 0.35 and 18 mM, and those for fructose were 1.6 and 25raM. The uptake of 0.2 mM glucose was inhibited by 2 mM fructoseand that of 2 mM fructose was inhibited by 2 mM glucose, butneither was inhibited by sucrose or sorbitol. O-methyl-glucose(OMG) also inhibited both the glucose and fructose uptakes.Therefore, the same transporter may mediate both glucose andfructose uptakes at lower concentrations; this hexose transportsystem differed from the sucrose and sorbitol transport systems. ¹Research Fellow of the Japan Society for the Promotion of Science. ²Present address: Faculty of Agriculture, Tohoku University,1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, 981 Japan.     

Effects of nitrogen nutrition on salt-stressed Nicotiana tabacum var. Samsun in vitro plantlets

Tobacco shoots were grown in vitro for 35 d, in MS culture mediummodified to include various sources (nitrate-N, ammonium-N ora mixture) and levels (0–120 mM) of N, and in the presenceof 0–180 mM NaCI or iso-osmotic concentrations of mannitol.Growth of control plantlets was significantly inhibited whenNH₄⁺-N was the sole N source, and at high (120 mM) NO^–₃-N supply. Under conditions of salt stress (90 and 180 mM NaCI)growth was repressed, with roots being more severely affectedthan shoots. Salinity also inhibited root emergence in vitro.The only alleviation of the salt stress by nitrate nutritionobserved in this study was on shoot growth parameters of plantletsgrown on 60 mM NO^–₃-N and 90 mM NaCI. Although both weresignificantly inhibited by NaCI, nitrate reduc-tase activitywas more severely affected than nitrate uptake. When mannitolreplaced NaCI in the culture medium, similar Inhibition of growth,nutrient uptake and enzyme activity were recorded. These observations,together with the relatively low recorded values for Na⁺ andCI^– uptake, indicate that under in vitro salt stress conditionsthe negative effects of NaCI are primarily osmotic. Key words: Growth, nitrogen metabolism, osmotic stress, salinity     

Regulation of IBA synthetase from maize (Zea mays L.) by drought stress and ABA

The rate of indole-3-butyric acid (IBA) synthesis in maize seedlingsis dependent on the culture conditions of the plants. When theseedlings were grown on filter paper soaked with different amountsof water, the activity of IBA synthetase differed strongly.High amounts of water (150 and 200 ml per bowl) inhibited IBAsynthesis completely in vitro, whereas 30 and 50 ml water perbowl increased the activity dramatically. Under conditions whereIBA synthetase was inhibited (150 ml H₂O), an increase of enzymeactivity was observed when abscisic acid (ABA) was exogenouslyadded in concentrations between 510^–4 to 510^–7M. Under ‘drought’ conditions (50 ml H₂O per bowl)the same ABA concentrations were inhibitory. Jasmonic acid andsalicylic acid also enhanced IBA synthetase activity to someextent, whereas indole-3-acetic acid (IAA) and kinetin had noeffect. Activity could also be enhanced by osmotic stress (NaCIand sorbitol), but not under temperature stress. In accompanyinginvestigations the endogenous contents of IAA, IBA, and ABAunder the different culture conditions have been determinedas well as the energy charge of the seedlings. Similar observationshave been made with Amaranthus, wheat and pea seedlings Key words: Abscisic acid, Amaranthus paniculatus, drought stress, inole-3-butyric acid biosynthesis, Pisum sativum, Triticum aestivum, Zea mays     

Calcium Chelator and Channel Blockers Suppress the IAA-Induced Membrane Hyperpolarization without Inhibiting the Following Growth Promotion in Hypocotyl Sections of Vigna unguiculata under Xylem Perfusion

The effects of the Ca²⁺ chelator EGTA and the Ca²⁺ channel blockersLa³⁺, Gd³⁺, nifedipine, and verapamil on the IAA-induced earlyresponses of hypocotyl sections of Vigna unguiculata were studiedto assess the involvement of Ca²⁺ in the IAA-induced hyperpolarizationfollowed by growth promotion using the xylem perfusion method.The IAA-induced hyperpolarization was distinctly inhibited bypretreatment with EGTA, La³⁺, and/or Gd³⁺ (5 mM each). EGTAinhibited by about 80–90% while La³⁺ and Gd³⁺ inhibitedby about 60–75%. The sustained hyperpolarization afterIAA application was also inhibited by these reagents. Nifedipineand verapamil (50 µM each) had little effect on the IAA-inducedhyperpolarization and growth promotion. The inhibitory effectsof EGTA, La³⁺ and Gd³⁺ on the membrane potentials were not dueto changes in a passive component, but mainly to decreases inan elec-trogenic component. The effect of EGTA was reversible,while those of La³⁺ and Gd³⁺ were not. Present results suggestthat the influx of Ca²⁺ through the plasma membrane might berequired not only for the initiation of hyperpolarization byIAA but also for the following sustained hyperpolarization andmay therefore play an essential role in the IAA-induced activationof the electrogenic proton pumps at the plasma membrane in Vignahypocotyls. Inhibition of the hyperpolarization by EGTA, La³⁺,and Gd³⁺, however, did not involve inhibition of the promotionof growth by IAA. It seems that activation of the electrogenicproton pumps at the plasma membrane is not always the indispensableprimary step of the IAA-induced promotion of elongation growth.Some alternative pathway of proton extrusion might take part. ³Present address: Laboratory of Biochemistry, Graduate SchoolofBioagricultural Sciences, Nagoya University, Nagoya, 464-8601Japan.     

Stimulatory Effect of Chloride Ions on NADP Malic Enzyme from Leaves of two C4 Species of Cyperus

NADP malic enzyme (EC 1.1.1.40 [EC] ) from leaves of two C₄ speciesof Cyperus (C. rotundus and C. brevifolius var leiolepis) exihibiteda low level of activity in an assay mixture that contained lowconcentrations of Cl^–. This low level of activity wasmarkedly enhanced by increases in the concentration of NaClup to 200 mM. Since the activity of NADP malic enzyme was inhibitedby Na₂SO₄ and stimulated by relatively high concentration ofTris-HCl (50–100 mM, pH 7–8), the activation ofthe enzyme by NaCl appears to be due to Cl^–. Variationsin the concentration of Mg²⁺ affected the K_A (the concentrationof activator giving half-maximal activation) for Cl^–,which decreased from 500 mM to 80 mM with increasing concentrationsof Mg²⁺ from 0.5 mM to 7 mM. The K_m for Mg²⁺ was decreased from7.7 mM to 1.3 mM with increases in the concentration of NaClfrom zero to 200 mM, although the increase of V_max was not remarkable.NADP malic enzyme from Cyperus, being similar to that from otherC₄ species, was able to utilize Mn²⁺. The K_m for Mn²⁺ was 5mM, a value similar to that for Mg²⁺. The addition of 91 mMNaCl markedly decreased the K_m for Mn²⁺ to 20 +M. NADP malicenzyme from Setaria glauca, which contains rather less Cl^–than other C₄ species, was inactivated by concentrations ofNaCl above 20 mM, although slight activation of the enzyme wasobserved at low concentrations of NaCl at pH7.6. (Received February 20, 1989; Accepted June 12, 1989)     

Energy Coupled Transport of Sorbitol and Other Sugars into the Protoplast Isolated from Apple Fruit Flesh

The uptake kinetics of sorbitol, sucrose, glucose and fructoseacross the plasma membrane using protoplasts isolated from applefruit flesh (Malus pumila Mill. var. domestica Schneid.) wasinvestigated. When sorbitol was taken up into the cell, PCMBS-sensitivesaturable transport was distinguishable from the diffusive transport.At a low sorbitol concentration, the saturable transport systemaccounted for more than 50% of the total uptake, whereas ata high concentration the diffusive transport system was moredominant. The saturable transport was suggested be a carrier-mediatedtransport system coupled with ATP because the system was inhibitedCCCP or orthovanadate. The K_m value for sorbitol was computedto be 3.6mM. A carrier-mediated transport system coupled withATP was also observed for glucose and fructose with correspondingK_m values of 5.0 and 2.5 mM. However, no saturable transportfor sucrose was observed over a range of 0.1 to 10 mM sucroseconcentration. The relationship among these sugar transportsystems across the plasma membrane, apoplastic unloading, andsugar accumulation vacuoles are discussed. ¹Present address: Laboratory of Horticulture, Faculty of Agriculture,Nagoya University, Chikusa, Nagoya 464, Japan. (Received April 8, 1988; Accepted June 8, 1988)     

Salt Stress Causes Acceleration of Purine Catabolism and Inhibition of Pyrimidine Salvage in Zea mays Root Tips

Nucleotide metabolism was studied in apical 5.0 mm root tipsof corn plants (Zea mays L., cv. Pioneer 3906) hydroponicallycultured for 7 d and then salinized for 19 d at a rate calculatedto reduce the osmotic potential (_o) of the solutions by O.1MPad^–1 to a final _o = -0.4 MPa. Saline treatments withtwo different molar ratios of Ca₂₊/Na⁺ were employed, viz.,0–03 (2.5 mol m^–3 CaCl₂ + 86.5 mol m^–3 NaCl)for the NaCl treatment and 0.73 (31.5 mol m^–3 CaCl₂ +43.1 mol m^–3 NaCl) for the NaCl + CaCl₂ treatment. Bothsalt treatments reduced root growth by more than 30%. The capacityof roots to provide purine nucleotides either by de novo synthesisor by re-utilization of existing bases, e.g. salvage of hypoxanthineto adenine nucleotides, was not affected by either salt treatment.However, catabolism of hypoxanthine was accelerated more than3.5-fold by both salt treatments, demonstrating an increasedcapacity for purine catabolism which would shift the normal1: 1 ratio of synthesis: degradation of purine nucleotides observedfor the roots of healthy control plants to less than 0.2 duringsalt stress. The ratio of pyrimidine nucleotide synthesis: degradationwas also reduced. In this case, the unfavourable shift towardnucleotide degradation resulted because both salt treatmentsreduced salvage capacity by more than 25%, but had no compensatingeffect on de novo synthesis or catabolism of pyrimidines. Key words: Salinity, osmotic potential, nucleotide metabolism     

Effect of osmotic shock on auxin-induced cell extension, cell wall changes and acidification in Avena coleoptile segments

The effect of plasma membrane alteration caused by osmotic shockof different strengths on the auxin-induced responses of Avenacoleoptile cells was observed. Osmotic shock brought about by0.5–0.7 M mannitol solution for 10 or 30 min, followedby phosphate-buffer (1 mM, pH 6.0) treatment for 10 min at 4?Ccaused no significant inhibition of auxin-induced cell extension.The osmotic shock did not affect auxin-induced cell wall looseningrepresented by stress-relaxation time and a decrease in thenoncellulosic glucose level of the cell wall. The shock causedonly a temporary inhibition of transmembrane potential and noinhibition of oxygen consumption. However, it inhibited auxin-stimulatedH⁺ secretion which was reversed by 0.1 mM CaCl₂. We concludedthat the Osmotic shock may partly modify the plasma membranerelated to the hydrogen ion pump which interacts with auxin,but this modification which is reflected little by the transmembranepotential and cellular metabolism, is not closely related toauxin-induced cell wall loosening and thus cell extension inAvena coleoptiles. ³ Present address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan (Received February 17, 1978; )     

The effects of valinomycin on respiration and volume changes in plant mitochondria

The effects of valinomycin on the respiration and volume changeshave been studied with isolated mitochondria from bean hypocotyl(Phaseolus vulgaris L.) and cauliflower bud (Brassica oleraceaL.). In the presence of 10 mM K salts of chloride, acetate,phosphate, and sulfate respiration is stimulated by valinomycinconcomitant with osmotic swelling. When swelling declines respirationwith organic acid substrates also declines. In the presenceof the K salts of acetate and PO₄ but not Cl^– the terminationof respiration leads to contraction. The contraction in K-PO₄is inhibited by addition to the external medium of between 65to 100 mM K-PO₄. The results are interpreted to suggest thatvalinomycin in the presence of KCl facilitated the movementof K down an electrical gradient, with the Cl anion followingand osmotic swelling resulting. However, in a medium containingacetate or PO₄ the anions are actively transported against anelectrical gradient at the expense of metabolic energy. Valinomycinfacilitates the influx of K⁺ with the actively transported anionand swelling follows. When respiration terminates the activelytransported anions move passively back down their electrochemicalgradient and osmotic contraction follows. ¹ Present address: Department of Biology, Fort Lewis College,Durango, Colorado 81301, U.S.A. (Received July 21, 1972; )     

A sulfite-dependent adenosine triphosphatase of Thiobacillus thiooxidans. Its partial purification and some properties

A sulfite-dependent ATPase [EC 3.6.1.3 [EC] ] of Thiobacillus thiooxidanswas activated and solubilized by treatment with trypsin [EC3.4.4.4 [EC] ], and purified 84-fold with a 32% recovery. It requiredboth Mg²⁺ and SO₃^2– for full activity, and its optimumpH was found at 7.5–8.0. Mn²⁺, Co²⁺, and Ca²⁺ could partiallysubstitute for Mg²⁺, while SeO₃^2– and CrO₄^2– couldpartially substitute for SO₃^2–. The enzyme hydrolyzed ATP and deoxy-ATP most rapidly and otherphosphate esters were poorer substrates. The apparent Km valuefor ATP was 0.33 mM. The enzyme activity was strongly inhibitedby 0.2 mM NaN₃ and 10 mM NaF. (Received July 27, 1977; )     

Effect of Ca2+ on Tonoplast Potential of Permeabilized Characeae Cells

Using permeabilized characean cells in which the ionic conditionsat the cytoplasmic side of the tonoplast are easily controlled,effects of Ca²⁺ ion on tonoplast potential were examined. Whenthe cell was treated with 1 µM Ca²⁺, the tonoplast potential(E_M became positive in a complicated manner in Chara corallinawhile it simply became negative in Nitella axilliformis. Whenthe cell was treated with 9-antracenecarboxylic acid, a Cl^–-channelinhibitor, E_m became more negative and the response of E_m toCa²⁺ was significantly suppressed. It is suggested that Ca²⁺activates Cl^–-channel at a low concentration and inactivatesat a higher one in C. corallina while it simply inactivate Cl^–-channelin N. axilliformis. ¹Present address: Biological Laboratory, The University of theAir, Wakaba 2-11, Wakaba, 260 Japan. (Received August 22, 1988; Accepted December 26, 1988)     

Induction of Light Dependent Pyruvate Transport into Chloroplasts of Mesembryanthemum crystallinum by Salt Stress

Mode of photosynthesis in Mesembryanthemum crystallinum changesfrom C₃ to Crassulacean acid metabolism (CAM) when the plantswere stressed with high salinity. [¹⁴C]Pyruvate uptake for 30s into intact chloroplasts isolated from leaves of the CAM modeof M. crystallinum was enhanced more than 5-fold in the lightcompared with that in the dark. The stromal concentration ofpyruvate in the light reached to more than 2.5 times of themedium. In contrast, little or no pyruvate uptake occurred inchloroplasts from C₃ leaves in either light or dark condition.The initial uptake rate (10 s incubation at 4°C) into theCAM chloroplasts in the light was about 3-fold higher than therate in the dark. K_m and V_max of the initial uptake in the lightwere 0.54 mM and 8.5 µmol (mg Chl)^–1 h^–1 respectively.These suggest that pyruvate was actively incorporated into theCAM chloroplasts against its concentration gradient across theenvelope in the light. When hydroponically grown M. crystallinumwere stressed by 350 mM NaCl, the capacity of chloroplasts forpyruvate uptake was induced in 6 d corresponding to the inductionof the activities of PEP-carboxylase and NAD(P)⁺-malic enzymesin response to salt stress. (Received October 12, 1995; Accepted January 19, 1996)     

Auxin Changes Both the Extensibility and the Yield Threshold of the Cell Wall of Vigna Hypocotyls

Elongation of plant stem is governed by two simultaneous processes:irreversible yielding of the cell wall and uptake of water.Among many candidates for the parameters that regulate and/or restrict growth, we focused on the mechanical propertiesof the cell wall and determined those parameters that governthe process of IAA-induced growth by means of the pressure-jumpmethod combined with the pressure-probe technique. The elongation growth of segments excised from the elongationzone of Vigna hypocotyls was accelerated by xylem perfusionwith 10^–4 M IAA. During the promotion of growth, boththe extensibility () of the cell wall and the effective turgor(Pⁱ–Y) increased while only a little or no change in theintracellular pressure (Pⁱ) occurred. These results indicate that IAA increases not only the extensibilityof the cell wall but also the effective turgor, i.e., the drivingforce for yielding of the cell wall. However, the driving forceis not increased by the increase in Pⁱ but by the decrease inthe yield threshold (Y). These results suggest that Y is adjustableduring the regulation of growth. ¹Present address: Department of Biology, Faculty of Science,Okayama University, Okayama, 700 Japan (Received September 20, 1990; Accepted November 27, 1990)     

Quantitative Analysis of ATP-Dependent H+ Efflux and Pump Current Driven by an Electrogenic Pump in Nitellopsis obtusa

Internodal cells of Nitellopsis were made tonoplast-free byperfusion with a medium containing EGTA. Cytoplasmic concentrationsof solutes were controlled by a second perfusion with mediaof known composition. The electrogenic pump current (I_p), whichwas calculated from electrical data obtained from cells withand without ATP, was compared with the current carried by H⁺(I_H⁺) across the plasma membrane. A close correlation betweenI_p and I_H⁺ was found under various internal and external conditions.(1) I_p and I_H⁺ depended on the internal ATP and showed Michaelis-Mententype saturation curves. For I_p, K_m was 120 µM and themaximum current V_max was 15.1 mA m^–2, while for I_H⁺, K_mwas 160 µM and V_max was 16.6 mA m^–2. (2) I_p andI_H⁺ showed almost the same I_H²⁺ dependence. The Mg²⁺-dependentI_p was 19.5 mA m^–2, while the Mg²⁺-dependent I_H²⁺ was17.7 mA m^–2. (3) I_H²⁺ was maximal at an external pH of8 and decreased both in acidic and alkaline pH ranges. I_p wasnearly equal to I_H⁺ in the pH range between 8 and 5. (4) I_H⁺became maximal at an internal pH of 7.3, which is nearly thesame as the pH for maximal electrogenecity found by Mimura andTazawa (1984). All these facts support the idea proposed in our previous paper(Takeshige et al. 1985) that the electrogenic ion pump locatedin the plasma membrane of Nitellopsis is the H⁺ pump. ¹ Dedicated to Professor Dr. Erwin Bünning on the occasionof his 80th birthday. (Received June 21, 1985; Accepted December 20, 1985)     

Abscisic Acid-Induced Membrane Potential Changes in Barley Aleurone Protoplasts: a Possible Relevance for the Regulation of rab Gene Expression

The effect of ABA on the membrane potential of barley (Hordeumvulgare cv. Himalaya) aleurone protoplasts was studied by measuringthe distribution of the lipophilic cation tetraphenylphosphonium(TPP⁺). The resting membrane potential (E_m) according to ourmeasurements with TPP⁺ is about –53 mV and is in agreementwith membrane potential values as measured with intracellularmicroelectrodes (about –55 mV). The TPP⁺-measurementscould demonstrate a clear dependence of the resting E_m on theexternal pH (pH_e). Stimulation of the protoplasts with ABA induced a transienthyperpolarization of the membrane to –62 mV as measuredwith TPP⁺. The hyperpolarization was ABA-concentration dependent. Inhibition of the H⁺-ATPases with the specific proton pump inhibitorsdiethylstilbestrol (DES) or Micanozole effectively preventedhyperpolarization. This indicates that the hyperpolarizationis consistent with the activation of plasma membrane H⁺-ATPases.The K⁺-inward rectifier inhibitor BaCl₂ was able to prolongthe hyperpolarization. This result suggests that the hyperpolarizationcauses the opening of K⁺-channels. The ABA-induced proton-pump activation may be involved in ABA-inducedgene-expression, as DES was able to inhibit this gene expression.BaCl₂ did only show a slight inhibitory effect on ABA-inducedgene-expression. (Received January 4, 1994; Accepted April 12, 1994)     

Multiple effects of the inhibitory protein of ethylene production on cellular metabolisms

The effects of an inhibitory protein of ethylene productionisolated from etiolated mung bean hypocotyls (Planta 113: 115,1973) were investigated. Etiolated mung bean hypocotyl segmentsincubated with IAA for 3 hr (1st incubation) to induce ethylene-producingactivity were incubated for 1 hr with IAA in the presence ofthe inhibitory protein and a radioactive material to measuremetabolic activity. Under the conditions where ethylene productionwas inhibited 80% or more by the protein, RNA synthesis, proteinsynthesis and phosphate uptake were suppressed 55–60,65–80, and 60–75%, respectively. Conversion of 1-¹⁴C-acetateto CO₂, lipid, basic and neutral fractions was also inhibited,but the degrees of inhibition were much less than those forthe other processes. When the segments pretreated with the inhibitoryprotein during the 1st incubation period were washed free ofthe protein and assayed for their metabolic activities, theinhibition of RNA and protein syntheses and of phosphate uptakewas partially restored, while ethylene-producing activity wasfully restored to the control level. Similar reversible inhibitoryeffects were also observed for those metabolic activities inthe tissue segments not treated with IAA, thus not producinginduced ethylene. Oxygen uptake and conversion of U-¹⁴C-glucoseto CO₂ were not affected by the inhibitory protein. The possibilitythat the inhibitory protein acts on cell surface membranes andthe modified membranes affect the regulatory mechanism of cellularmetabolism is discussed. ¹ This investigation was supported in part by grants from theMinistries of Education (B-248009), and of Agriculture and Forestryof Japan. (Received November 4, 1977; )