A Possible Regulation of Ethylene Production by the Epidermis in Mung Bean Hypocotyl Sections

IAA-induced and l-aminocyclopropane-l-carboxylic acid (ACC)-dependentethylene production in etiolated mung bean (Vigna radiata [L]Wilczek) hypocotyl sections does not occur in epidermal cells(Todaka and Imaseki 1985). Mung bean hypocotyls contain a proteinwhich inhibits auxin-induced ethylene biosynthesis in hypocotylsections (Sakai and Imaseki 1975a, b). This inhibitory proteinwas also found to inhibit ACC-dependent ethylene productionin hypocotyl sections, but not in hypocotyl sections from whichthe epidermis had been removed. Uptake of ACC by both unpeeledand peeled sections was not inhibited by the protein. Similarly,IAA-induced ethylene production was inhibited by the proteinin unpeeled hypocotyl sections, but not in peeled sections.The protein was not inactivated in peeled sections, as proteinsynthesis by peeled sections was inhibited to the same extentas in unpeeled sections. The protein inhibited incorporationof 3,4-[¹⁴C]-methionine into ACC and ethylene in unpeeled sections,but not in peeled sections, whereas oxidation of the labeledmethionine into CO₂ was inhibited by the protein to a similarextent in both types of hypocotyl sections. KCN, a potent inhibitorof ethylene production, inhibited both IAA-induced and ACC-dependentethylene production in both peeled and unpeeled hypocotyl sections.It is likely that the epidermis plays some role in controllingethylene production which occurs in stem cells other than epidermalcells. (Received July 16, 1985; Accepted October 21, 1985)     

On the Relationship between Extracellular pH and the Growth of Excised Pea Stem Segments

Studies with stem segments of peas (Pisum sativum L. var. Alaska) suggest that the pH of the medium bathing elongating tissue does not always reflect intramural (cell wall) conditions or that pH is not a controlling factor in elongation. Peeled, green segments, and peeled or nonpeeled etiolated segments appear to regulate the pH of their bathing medium causing it to become acidified with or without the addition of auxin. The growth rates of segments are greatest during a period before acidification is evident and slow during the time in which the medium becomes acidified. We cannot reproduce the dramatic auxin-induced pH shifts reported in the literature because the control segments are becoming more acid also; but there is some evidence that acidification may occur in response to auxin treatments. K⁺ additions mimic the acidifying tendency of auxin but are without growth-promoting effect. Emergent growth (an extremely rapid burst of growth following anaerobic treatments) is not accompanied by a drop in pH of the bathing medium. Proper aeration of the bathing medium in extracellular pH studies is crucial and may explain differences between our results and other published accounts. The data suggest that the techniques used for most extracellular pH studies may not very closely approximate in vivo conditions or properly reflect intramural H⁺ concentration fluxes.     

pH-Dependence of Extension Growth in Avena Coleoptiles and Its Implications for the Mechanism of Auxin Action

The pH-dependence of acid-induced growth in excised segments of Avena sativa coleoptiles has been reinvestigated in the pH range 3 to 7. In contrast to previous reports (e.g. DL Rayle [1973] Planta 114: 63-73), only acidic buffers with a pH below 5.0 induce an extension response. A pH of 3.5 to 4.0 is required to mimic auxin-mediated growth. Very similar pH-response curves are obtained with both intact (abraded) and peeled coleoptiles. These results agree with the recent finding of a similarly low sensitivity to protons in maize coleoptiles. It is shown that the apparently much higher sensitivity to protons previously reported for peeled Avena coleoptiles is due to incubating the tissue in buffer of pH 6.8 between peeling and measuring the effect of acidic buffers. Neutral pH reversibly inhibits the spontaneous extension burst originating on release from tissue tension after removing the epidermis. Reversal of this inhibition can be achieved by buffers of pH 5.0 to 6.0 (or distilled water), thereby simulating an acid-induced growth response in this pH range. It is concluded that true acid-induced wall-loosening generally does not take place above pH 5.0 and that a pH considerably below 4.0 is required in order to stimulate growth to an extent comparable to that obtained in response to auxin. The “acid-growth theory,” which requires an acid-mediated loosening of the cell wall in the pH range 5 to 6, this pH being established by auxin-induced proton excretion, can therefore also not be substantiated in Avena.     

Reversion from light-induced inhibition of seed germination by respiratory inhibitors

Treating of the dark-imbibed lettuce (Lactuca sativa L.) seeds prior to light irradiation with 1 millimolar KCN or NaN₃ in the dark for 3 hours prevented blue light and far-red light-induced inhibitions of phytochrome-mediated germination. Similarly, salicylhydroxamic acid (SHAM) at 10 millimolar counteracted the blue and far-red light inhibitions, the combined application of KCN and SHAM being more effective than KCN or SHAM alone in some experiments. These respiratory inhibitors slightly inhibited phytochrome-mediated lettuce seed germination. These results indicate that both CN-sensitive, conventional cytochrome oxidase and CN-resistant (SHAM-sensitive), alternative respiration may be involved in the light inhibition or that an appropriate balance of both may be necessary for the light inhibition.     

Effect of indoleacetic Acid and hydroxyproline on isoenzymes of peroxidase in wheat coleoptiles

Indoleacetic acid at 0.017 millimolar inhibited the formation of three peroxidase isoenzymes in both soluble and wall-bound enzyme fractions of wheat coleoptile (Triticum vulgare) tissue. Hydroxyproline at 1 millimolar prevented the indoleacetic acid-induced inhibition. Indoleacetic acid oxidase activity in the soluble fraction was decreased by indoleacetic acid and was restored by hydroxyproline. Most of the indoleacetic acid oxidase activity was located in the electrophoretic zones occupied by two of the peroxidase isoenzymes influenced by indoleacetic acid and hydroxyproline. At least part of the effect of hydroxyproline on auxin-induced elongation of coleoptile tissue may be through control of auxin levels by indoleacetic acid oxidase.     

Auxin-induced growth of Avena coleoptiles involves two mechanisms with different pH optima

Although rapid auxin-induced growth of coleoptile sections can persist for at least 18 hours, acid-induced growth lasts for a much shorter period of time. Three theories have been proposed to explain this difference in persistence. To distinguish between these theories, the pH dependence for auxin-induced growth of oat (Avena sativa L.) coleoptiles has been determined early and late in the elongation process. Coleoptile sections from which the outer epidermis was removed to facilitate buffer entry were incubated, with or without 10 micromolar indoleacetic acid, in 20 millimolar buffers at pH 4.5 to 7.0 to maintain a fixed wall pH. During the first 1 to 2 hours after addition of auxin, elongation occurs by acid-induced extension (i.e. the pH optimum is <5 and the elongation varies inversely with the solution pH). Auxin causes no additional elongation because the buffers prevent further changes in wall pH. After 60 to 90 minutes, a second mechanism of auxin-induced growth, whose pH optimum is 5.5 to 6.0, predominates. It is proposed that rapid growth responses to changes in auxin concentration are mediated by auxin-induced changes in wall pH, whereas the prolonged, steady-state growth rate is controlled by a second, auxin-mediated process whose pH optimum is less acidic.     

Inhibition of Low pH-induced Elongation in Avena Coleoptiles by Abscisic Acid

An angular position-sensing transducer was used to make continuous measurements of acid-induced elongation of Avena sativa coleoptile segments. Elongation rates at pH 4.5 (5 mm succinate buffer) were about 5-fold greater than those at pH 6.0. Buffered 0.1 mm abscisic acid produced a partial decrease of the growth rate. Pretreatments with abscisic acid buffered at pH 6.0 usually caused a further reduction of the elongation response when the coleoptile segments were subsequently placed in buffer at pH 4.5 containing abscisic acid. Abscisic acid did not completely prevent the pH effect in any of these experiments, and the brief latent period of the pH response was not affected by abscisic acid treatments. At pH 4.5, where the inhibitory effect of ABA was maximum, low pH-induced elongation was also inhibited by KCN and HgCl₂. These results suggest that pH-(4.5) induced elongation in this system may be dependent on some metabolic processes and that abscisic acid-induced inhibition of this elongation may involve an interaction with these processes.     

Auxin-induced hydrogen-ion secretion in Avena coleoptiles and its implications

Summary The dose response curve for hydrogen-ion-induced extension growth in Avena coleoptile segments has been reinvestigated. The previously published optimum (pH 3.0) is in error by about two orders of magnitude. The correct optimum is around pH 5.0. This discrepancy is thought to be due to the impermeable nature of the cuticle to hydrogen ions. In the present study the cuticular barrier to H⁺ entry was circumvented by using coleoptile segments from which the epidermis with cuticle were physically removed. Using such peeled coleoptile sections, it was also found that auxin can rapidly (20–30 min) initiate H⁺ secretion and that the magnitude of auxin-induced secretion is sufficient to initiate considerable cell-extension growth. Furthermore, it is shown that the secretion response is specific for active auxins, and inhibited by agents which inhibit auxin-induced growth (dinitrophenol, abscisic acid, cycloheximide, valinomycin and others). These results make it very likely that H⁺ secretion is responsible, at least in part, for the initiation of auxin-induced cell wall loosening and extension growth.     

Invertases in Oat Seedlings: SEPARATION, PROPERTIES, AND CHANGES IN ACTIVITIES IN SEEDLING SEGMENTS

The soluble invertase activity in etiolated Avena seedlings was highest at the apex of the coleoptile and much lower in the primary leaf, mesocotyl, and root. The activity in all parts of the seedling consisted of two invertases (I and II) which were separated by chromatography on diethylaminoethylcellulose. Both enzymes appeared to be acid invertases, but they differed in molecular size, pH optimum, and the kinetic parameters K_m and V_max of their action on sucrose, raffinose, and stachyose. Invertase II had low stability at pH 3.5 and below, and exhibited high sensitivity to Hg²⁺, with complete inhibition by 2 micromolar HgCl₂. Segments of coleoptiles incubated in water lost about two-thirds of the total invertase activity after 16 hours. The loss of activity was due primarily to a decrease in the level of invertase II. The loss of invertase was decreased by indoleacetic acid, 2,4-dichlorophenoxyacetic acid, and α-naphthaleneacetic acid but not by β-naphthaleneacetic acid and p-chlorophenoxyisobutyric acid. Conditions that inhibited auxin-induced growth of the segments (20 millimolar CaCl₂ and 200 millimolar mannitol) also blocked the auxin effect on invertase loss.     

Effect of inorganic cations and metabolic inhibitors on putrescine transport in roots of intact maize seedlings

The specificity and regulation of putrescine transport was investigated in roots of intact maize (Zea mays L.) seedlings. In concentration-dependent transport studies, the kinetics for putrescine uptake could be resolved into a single saturable component that was noncompetitively inhibited by increasing concentrations of Ca²⁺ (50 micromolar to 5 millimolar). Similarly, other polyvalent cations, including Mg²⁺ (1.8 millimolar) and La³⁺ (200 micromolar), almost completely abolished the saturable component for putrescine uptake. This suggests that putrescine does not share a common transport system with other divalent or polyvalent inorganic cations. Further characterization of the putrescine transport system indicated that 0.3 millimolar N-ethyl-maleimide had no effect on putrescine uptake, and 2 millimolar p-chloromercuribenzene sulfonic acid only partially inhibited transport of the diamine (39% inhibition). Metabolic inhibitors, including carbonylcyanide-m-chlorphenylhydrazone (20 micromolar) and KCN (0.5 millimolar), also partially inhibited the saturable component for putrescine uptake (V_max reduced 48-60%). Increasing the time of exposure to carbonylcyanide-m-chlorphenylhydrazone from 30 minutes to 2 hours did not significantly increase the inhibition of putrescine uptake. Electrophysiological evidence indicates that the inhibitory effect on putrescine uptake by these inhibitors is correlated to a depolarization of the membrane potential, suggesting that the driving force for putrescine uptake is the transmembrane electrical potential across the plasmalemma.     

Effect of sugars and amino acids on membrane potential in two clones of sugarcane

Sugarcane (Saccharum officinarum L.) leaf parenchyma cells bathed in 1X solution maintained an average membrane potential of −135 millivolts in the dark. No difference in membrane potential was found between clones 51 NG 97 and H50 7209. An electrogenic pump appears to contribute to membrane potential in these cells. Sugars (25 millimolar) added externally caused the following membrane potential depolarizations (in millivolts) in clone 51 NG 97: glucose, 18 ± 4; galactose, 24 ± 7; 3-O-methylglucose, 10 ± 4; sucrose, 22 ± 3; fructose, 21 ± 7; raffinose, 9 ± 3; mannitol, 0; lactose, 0; melibiose, 0; and 1-O-methyl-α-galactose, 0. Glycine (25 millimolar) and serine (10 millimolar) caused depolarizations of 47 ± 7 and 23 ± 2 millivolts, respectively. Depolarization shows saturation kinetics with respect to glucose concentration, with a K_m of 3 to 6 millimolar. The metabolic inhibitors KCN and salicyl hydroxamic acid together caused depolarization of the membrane potential and greatly inhibited depolarization by 25 millimolar glucose and 25 millimolar raffinose. In a series of substitution experiments, glucose (25 millimolar) caused almost total inhibition of depolarization by raffinose, sucrose, and 3-O-methylglucose (all 25 millimolar), but only partial inhibition of depolarization to 25 millimolar glycine. Glycine (25 millimolar), also, only partially inhibited depolarization by 25 millimolar glucose. Total depolarization to 25 millimolar glycine and 25 millimolar glucose was comparable to the amount of depolarization of membrane potential caused by 1 millimolar KCN plus 1 millimolar salicyl hydroxamic acid. The results are consistent with a co-transport mechanism of membrane transport, with sugars and amino acids being transported by separate carrier systems.     

Effects of Respiration Inhibitors and Uncouplers on Dark- and Light-Induced Leaflet Movements of Cassia fasciculata

Respiration inhibitors, in particular KCN and NaN₃, inhibited slightly the dark-induced (scotonasty) as well as the light-induced (photonasty) leaflet movements of Cassia fasciculata: they act only at concentrations higher than 1 millimolar and 0.1 millimolar, respectively. Amytal induced a stronger inhibitory effect on scotonasty. Salicylhydroxamic acid, which inhibits the cyanide-insensitive respiration pathway, was also poorly effective when applied alone. KCN and salicylhydroxamic acid applied together increased the inhibition. Uncouplers of oxidative phosphorylation were very effective: 2,4-dinitrophenol and carbonylcyanide-m-chlorophenylhydrazone inhibited the scotonastic movements at concentrations higher than 10 μm and 1 μm, respectively. Although uncouplers reduced the photonastic movements at higher concentrations, they promoted leaflet opening at other concentrations in an unexpected way.     

The Epidermis of the Pea Epicotyl Is Not a Unique Target Tissue for Auxin-Induced Growth

Previous research has suggested that the epidermis of dicotyledonous stems is the primary site of auxin action in elongation growth. We show for pea (Pisum sativum L.) epicotyl sections that this hypothesis is incorrect. In buffer (pH 6.5), sections from which the outer cell layers were removed (peeled) elongated slowly and to the same extent as intact sections. Addition of 10 micromolar indoleacetic acid to this incubation medium caused peeled sections to grow to the same extent and with the same kinetics as auxin-treated nonpeeled sections. This indicates that both epidermis and cortical tissues have the ability to respond rapidly to auxin and that the epidermis is not the sole site of auxin action in dicotyledonous stems. Previous reports that peeled pea sections respond poorly to auxin may have resulted from an acid extension of these sections due to the use of distilled water as the incubation medium.     

The effect of respiratory inhibitors on the fermentative ability of Pichia stipitis,Pachysolen tannophilus and Saccharomyces cerevisiae under various conditions of aerobiosis

Summary The growth and ethanol production by the d-xylose-fermenting yeasts Pichia stipitis and Pachysolen tannophilus under various conditions of aerobiosis responded similarly to the addition of the respiratory inhibitors potassium cyanide (KCN), antimycin A (AA), sodium azide and rotenone. However, the d-glucose-fermenting yeast Saccharomyces cerevisiae differed markedly from these yeasts in response to the inhibitors. In general the growth of the d-xylose-fermenting yeasts was inhibited by the respiratory inhibitors while ethanol production was either stimulated (especially when oxygen was available) or unaffected or inhibited by rotenone or AA or KCN and sodium azide, respectively. However, by exception KCN and AA stimulated ethanol production under aerobic conditions by Pichia stipitis and Pachysolen tannophilus respectively. Stimulatory or inhibitory effects by respiratory inhibitors were less marked in S. cerevisiae. These data suggest that unimpaired mitochondrial function is necessary for growth on d-xylose and optimal d-xylose fermentation. A requirement for membrane generated energy during d-xylose utilisation is indicated by 2,4-dinitrophenol inhibition of growth and fermentation.     

Effects of KCN and Salicylhydroxamic Acid on the Root Respiration of Pea Seedlings

Polarography, using cylindrical platinum electrodes, proved suitable for measuring changes in the internal apical O₂ concentration of the primary root of pea (Pisum sativum L. cv Meteor) effected by KCN and/or salicylhydroxamic acid (SHAM) in the bathing medium. An electrical rootaeration analog was used to help evaluate some of the results. Concentrations of KCN ≤0.05 millimolar had no significant effect. In response to 0.1 millimolar KCN, the O₂ concentration rose substantially for approximately 2 hours, then declined, and after 10 hours had frequently fallen below the pretreatment level. Such changes suggest an initial inhibition of cytochrome oxidase-mediated O₂ uptake followed by an induction of the alternative, cyanide-resistant respiratory pathway. These treatments proved nonlethal. Changes in O₂ concentration similar to those described for 0.1 millimolar KCN were observed in response to 1 and 10 millimolar KCN but these treatments were lethal and the root apex became soft and often appeared flooded. Roots survived and showed no significant responses when treated with SHAM at concentrations ≤5 millimolar. However, when the alternative pathway had been (apparently) induced by 0.1 millimolar KCN, the addition of 5 millimolar SHAM to the bathing medium caused a substantial and persistent rise in the root apical O₂ concentration, suggesting that this (nonlethal) concentration of SHAM could indeed inhibit O₂ uptake via the cyanide-resistant pathway.

It is concluded that while O₂ uptake normally occurs by the cytochrome pathway in the primary pea root, the alternative, cyanide-resistant pathway can be induced by 0.1 millimolar KCN.

     

Biphasic response of cucumber hypocotyl sections to auxin

The pattern of the response to auxin of cucumber hypocotyl sections,its relation to acid-induced growth and the role of the epidermiswere investigated. In longterm incubation, IAA had practicallyno effect on sections without epidermis (peeled sections). Timecourse measurementsshowed that the response to IAA of nonpeeledsections was biphasic; the first phase started right after IAAtreatment and continued for about 1 hr, then after a periodof 1 hr with a greatly decreased growth rate the second phasestarted with a higher rate. Peeled sections lacked thesecondphase; their growth rate in the first phase depended on theacidity of the incubation medium. These results were confirmedby utilizing one side-peeled sections and measuring the curvatureof the sections which represented the difference in the growthrate between the peeled and nonpeeled sides. The following pointswere suggested. The first phase resembles acid-induced growthand the growth capacity is limited by the epidermis; the secondphase is specific to auxin and requires the presence of theepidermis. The first phase was hardly influenced by temperature,while the second one was greatly affected; its Q,₁₀ being morethan 4.0, suggesting that the first phase is of physical nature. (Received December 26, 1975; )     

Reevaluation of the cyanide resistance of seed germination

Although high levels of KCN (53 micromoles per gram fresh weight of seed, corresponding to 3.2 millimolar) failed to block germination of lettuce seeds incubated in covered Petri dishes, the same levels totally blocked germination in sealed dishes. Inhibition was reversed by removing the seal. Placement of KCN remote from seeds also blocked germination in closed systems. Cyanide effectiveness was enhanced by acidifying the KCN solution but negated by the presence of a trap containing strong alkali. Low levels of aqueous HCN (2.6 micromoles HCN per gram, corresponding to 0.16 millimolar) injected into sealed dishes gave maximal inhibition of germination, suggesting that the effectiveness of KCN was due to formation of HCN in KCN solutions. Studies with nine additional crop species generally supported the interpretation that cyanide inhibition of germination has been underestimated in the past due to escape of volatile HCN from open systems.     

The Role of Protons in the Auxin-induced Root Growth Inhibition - A Critical Reexamination

According to the acid growth theory of auxin action, it has been proposed that auxin decreases root growth by inhibiting the proton pump, thus causing an alkalinization of the apoplast. This paper critically tests this hypothesis with corn (Zea mays L.) roots. It was found that: i) the pH-growth curve for roots exhibits a broad optimum ranging from pH 4.5 to 9. ii) Any acid-induced growth is of very short duration, iii) The low sensitivity of root growth to external pH is independent of both the pump activity and the buffer capacity of the bathing solution, iv) Neither incubation in acidic buffer nor stimulation of the proton pump reverts the auxin-induced root growth inhibition. It is concluded that the auxin-induced root growth inhibition is not mediated by cell wall alkalinization.     

Regulation of Apoplastic pH in Source Leaves of Vicia faba by Gibberellic Acid

Leaf discs of broad bean (Vicia faba L.), peeled on the spongy mesophyll side, rapidly altered the pH of the surrounding medium (apoplast). Using pH indicator paper appressed against the leaf, immediately after peeling, initial apoplastic pH was estimated to be 4.5. Changes in the apoplastic pH were measured with a microelectrode placed into a 100-microliter drop of an unbuffered solution (2 millimolar KCl, 0.5 millimolar CaCl₂, and 200 millimolar mannitol) on the peeled surface. Discs acidified the medium until the pH stabilized at about 5.0 (about 10 minutes). Acidification was inhibited by 50 micromolar sodium vanadate, an inhibitor of the plasmalemma H⁺-ATPase and attenuated by omitting the osmoticum or potassium ions from the medium. Fusicoccin (10 micromolar) greatly enhanced the rate of acidification. The presence of 0.1 to 1 micromolar gibberellic acid resulted in a slower rate of medium acidification. Gibberellic acid appeared to modulate the activity of the H⁺-translocating ATPase located at the plasma membrane of the mesophyll cells.     

The antagonism of IAA-induced hydrogen ion extrusion and coleoptile growth by diclofop-methyl

Diclofop-methyl (DM) (ester) was readily absorbed by peeled and unpeeled coleoptiles of wheat, Triticum aestivum L. cv. Waldron, and oat, Avena sativa L. cv. Garry. Substantial absorption of diclofop (acid) occurred only in peeled coleoptiles of the two species. IAA-induced acidification in peeled coleoptiles of both species was inhibited by 100 μ M DM or diclofop (acid) during a 3 to 4 h period. There was no recovery of acidification after DM or diclofop inhibition in oat coleoptiles; however, acidification in wheat coleoptiles recovered from inhibition by DM but not from diclofop. The recovery from DM inhibition may be due to a reduction in the diclofop pool derived from DM by efflux and metabolism (detoxification) in peeled wheat coleoptiles. Diclofop was not detoxified in oat coleoptiles. IAA-induced elongation of unpeeled oat coleoptiles was inhibited totally by 100 μ M DM but not by 100 μ M diclofop after 3.3 h of treatment. Wheat coleoptile elongation was relatively unaffected by either DM or diclofop. Basal elongation (no IAA) of both wheat and oat coleoptiles was inhibited by DM and diclofop. The inhibition by DM appeared to be irreversible, whereas the inhibition by diclofop was overcome by the addition of 10 μ M IAA.