Cooperation of Ethylene and Auxin in the Growth Regulation of Rice Coleoptile Segments

Elongation of coleoptile segments, having or not having a tip,excised from rice (Oryza sativa L. cv. Sasanishiki) seedlingswas promoted by exogenous ethylene above 0.3 µl l^–1as well as by IAA above 0.1 µM. Ethylene production ofdecapitated segments was stimulated by IAA above 1.0µM,and this was strongly inhibited by 1.0 µM AVG. AVG inhibitedthe IAA-stimulated elongation of the decapitated segment witha 4 h lag period, and this was completely recovered by ethyleneapplied at the concentration of 0.03 µl l^–1, whichhad no effect on elongation without exogenous IAA. The effectsof IAA and ethylene on elongation were additive. These factsshow that ethylene produced in response to IAA promotes ricecoleoptile elongation in concert with IAA, probably by prolongingthe possible duration of the IAA-stimulated elongation, butthat they act independently of each other. Moreover, AVG stronglyinhibited the endogenous growth of coleoptile segments withtips and this effect was nullified by the exogenous applicationof 0.03 µl l^–1 ethylene. These data imply that theelongation of intact rice coleoptiles may be regulated cooperativelyby endogenous ethylene and auxin in the same manner as foundin the IAA-stimulated elongation of the decapitated coleoptilesegments. Key words: oryza sativa, Ethylene, Auxin, Coleoptile growth     

Mechanism of cytokinin action on auxin-induced ethylene production

The stimulative effect of cytokinin on ethylene production hasbeen examined in etiolated mungbean hypocotyl segments. Therate of auxin-induced production linearly increased with timein a certain range of exogenous IAA concentration. The rateof induced production doubled with a 10-fold increase in exogenousIAA concentration or addition of benzyladenine at 5 µM.Benzyladenine did not suppress inactivation of the induced ethyleneproducing system. Although the free IAA level within the tissueswas slightly increased by benzyladenine accompanied by a decreasein IAAsp formation, the increased free IAA level did not accountfor the doubling of the production rate. When the tissues werepreincubated with benzyladenine alone followed by postincubationwith auxin, the rate of induced production in pretreated tissueswas significantly higher than that in untreated or buffer-treatedtissues supplied with auxin and benzyladenine simultaneouslyin the post-incubation medium. Formation and disappearance ofthe cytokinin effect were temperature dependent. The rate ofendogenous production was constant over an experimental periodand benzyladenine simply enhanced the rate several-fold aftera lag period. Kinetics of the cytokinin stimulation was notthe inductive type. Based on these observations, a possiblemechanism of the stimulative effect of cytokinin was discussed. ¹This research was partly supported by grants from the Ministryof Education of Japan (C-856043 and C-956037) and the AsahiPress. (Received May 7, 1975; )     

Role of oxygen in auxin-induced ethylene production

Ethylene production by IAA-treated mung bean hypocotyl segmentsunder various oxygen levels in the ambient atmosphere was examined.Rate of ethylene production was dependent upon oxygen levels,and gave a sigmoidal curve against oxygen levels. Tissue segmentspreincubated with IAA in low oxygen levels (1–10% O₂ inN₂) produced ethylene without a lag period at a rate higherthan that by control tissue segments preincubated in air, whenthey were exposed to a high oxygen level (air, 21% O₂). Theeffect of cycloheximide on tissue segments transferred froma low oxygen level to air was not much different from that onethylene production by control tissue segments previously incubatedin air. Incorporation of U-¹⁴C-leucine into the protein fractionby tissue segments placed in nitrogen was negligible, but thatin 2% oxygen was 10 to 14% of that in air. It was concluded that oxygen was an essential factor for boththe induction process of the ethylene producing system and thesynthesis of ethylene, and that although synthesis of ethyleneis dependent upon oxygen levels, formation of the ethylene producingsystem proceeded even under low oxygen levels. (Received January 13, 1977; )     

Auxin and Ethylene Control of Growth in Seedlings of Zea mays L. and Avena sativa L

The effects of applied ethylene on the growth of coleoptilesand mesocotyls of etiolated monocot seedlings (oat and maize)have been compared with those on the epicotyl of a dicot seedling(the etiolated pea). Significant inhibition of elongation by ethylene (10 µll^–1for 24 h) was found in intact seedlings of all three species,but lateral expansion growth was observed only in the pea internodeand oat mesocotyl tissue. The sensitivity of the growth of seedlingparts to ethylene is in the decreasing order pea internode,oat coleoptile and oat mesocotyl, with maize exhibiting theleast growth response. Although excised segments of mesocotyland coleoptile or pea internode all exhibit enhanced elongationgrowth in IAA solutions (10^–6–2 ? 10^–5 moll^–1), no consistent effects were found in ethylene. Ethyleneproduction in segments was significantly enhanced by applicationof auxin (IAA, 10^–5 mol l^–6 or less) in all tissuesexcept those of the eat mesocotyl. Segments of maize show a slow rate of metabolism of applied[2-¹⁴C]IAA (30 per cent converted to other metabolites within9 h) and a high capacity for polar auxin transport. Ethylene(10 µl l^–1 for 24 h) has little effect on eitherof these processes. The oat has a smaller capacity for polartransport than maize and the rate ef metabolism of auxin isas fast as in the pea (90 per cent metabolized in 6 h). Althoughethylene pretreatment does not change the rate of auxin metabolismin oat, there is a marked reduction in auxin transport. It is proposed that the insensitivity of maize seedlings toethylene is related to the supply and persistence of auxin whichcould protect the seedling against the effects of applied orendogenously produced ethylene. Although the mesocotyl of oatis sensitive to applied ethylene it may be in part protectedagainst ethylene in vivo by the absence of an auxin-enhancedethylene production system. The results are discussed in relationto a model for the auxin and ethylene control of cell growthin the pea.     

Auxin and Ethylene-stimulated Adventitious Rooting in Relation to Tissue 9

We have previously shown that both endogenous auxin and ethylenepromote adventitious root formation in the hypocotyls of derootedsunflower (Helianthus annuus) seedlings. Experiments here showedthat promotive effects on rooting of the ethylene precursor,1-aminocyclopropane-l-carboxylic acid (ACC) and the ethylene-releasingcompound, ethephon (2-chloro-ethylphosphonic acid), dependedon the existence of cotyledons and apical bud (major sourcesof auxin) or the presence of exogenously applied indole-3-aceticacid (IAA). Ethephon, ACC, aminoethoxyvinylglycine (an inhibitorof ethylene biosynthesis), and silver thiosulphate (STS, aninhibitor of ethylene action), applied for a length of timethat significantly influenced adventitious rooting, showed noinhibitory effect on the basipetal transport of [³H]IAA. Theseregulators also had no effect on the metabolism of [³H]IAA andendogenous IAA levels measured by gas chromatography-mass spectrometry.ACC enhanced the rooting response of hypocotyls to exogenousIAA and decreased the inhibition of rooting by IAA transportinhibitor, N-1-naphthylphthalamic acid (NPA). STS reduced therooting response of hypocotyls to exogenous IAA and increasedthe inhibition of rooting by NPA. Exogenous auxins promotedethylene production in the rooting zone of the hypocotyls. Decapitationof the cuttings or application of NPA to the hypocotyl belowthe cotyledons did not alter ethylene production in the rootingzone, but greatly reduced the number of root primordia. We concludethat auxin is a primary controller of adventitious root formationin sunflower hypocotyls, while the effect of ethylene is mediatedby auxin. Key words: Auxin, ethylene, adventitious rooting, sunflower     

Stimulation of Auxin Induced Ethylene Production in Mung Bean Hypocotyl Segments by 5,5' -Dithiobis-2-nitrobenzoic Acid

The non-permeant protein inhibitor 5,5'-dithiobis-2-nitrobenzoicacid (DTNB) was tested for its effects on auxin induced ethyleneproduction. There was a stimulation in the rate of auxin inducedethylene production at all concentrations of DTNB tested (1,2, 5, and 10 mM). The 5 mM DTNB treatment promoted the maximumstimulation of ethylene production with no further enhancementat the 10 mM concentration. After 12 hr ethylene productionplateaued with 0.1 mM indoleacetic acid (IAA) alone and in combinationwith 1 and 2 mM DTNB. Although the DTNB treatments plateauedit was at a higher level than IAA alone. Both the 5 and 10 mMtreatments of DTNB plus IAA did not show this leveling responseeven after 22 hr at which time these treatments were between90 and 100% higher than the control. There was no stimulationof ethylene production by DTNB in the absence of IAA. Segmentstreated with 10^–4 M rß-naphthaleneacetic acid(NAA) produced significantly higher levels of ethylene thanIAA at the same concentration. Stimulation of ethylene productionby DTNB was greatest at lower concentrations of IAA and NAA.The uptake of ¹⁴C-NAA by mung bean segments was 6-fold greaterin the presence of DTNB than in its absence. Ca^SS was requiredin the incubating media for DTNB to be effective. In the presenceof Ca^SS there was a highly significant increase in ethyleneproduction while in its absence there was no significant effect.The stimulation of IAA induced ethylene production appearedto have a pH optima of 4.6, at higher pH values this responsewas not shown. ¹ Approved for publication May 28, 1981 as paper number 6243in the journal series of the Pennyslvania Agricultural ExperimentStation. (Received June 10, 1981; Accepted January 5, 1982)     

The spontaneous growth response of maize coleoptile segments and the change in tissue 8

Decapitated segments from maize (Zea mays L.) coleoptiles orientedvertically in an upright position show a strong spontaneousgrowth response (SGR) 3 h after decapitation. The latent periodof the SGR is markedly reduced when these segments are orientedin an inverted position. Coleoptile segments with intact tipsexhibit a weak and transient SGR in the vertical upright orientation.However, in the inverted orientation, these segments show atypical SGR. The data are inconsistent with the current hypothesisthat the SGR is caused by a time-dependent increase in tissuesensitivity to auxin. The parallel increase in membrane potentialdifference and growth rate during the time-course of the SGRindicates a possible role for PM H⁺-ATPase in the establishmentof the SGR in maize coleoptile segments. Key words: Auxin, spontaneous growth response, membrane potential, plasma membrane H⁺-ATPase, Zea mays L.     

Mechanism of Auxin-induced Ethylene Production

Indoleacetic acid-induced ethylene production and growth in excised segments of etiolated pea shoots (Pisum sativum L. var. Alaska) parallels the free indoleacetic acid level in the tissue which in turn depends upon the rate of indoleacetic acid conjugation and decarboxylation. Both ethylene synthesis and growth require the presence of more than a threshold level of free endogenous indoleacetic acid, but in etiolated tissue the rate of ethylene production saturates at a high concentration and the rate of growth at a lower concentration of indoleacetic acid. Auxin stimulation of ethylene synthesis is not mediated by induction of peroxidase; to the contrary, the products of the auxin action which induce growth and ethylene synthesis are highly labile.     

Growth Regulators in Populus tremula

The seasonal variation in capacity to form suckers and in auxin level in bark and wood was determined in root segments of aspen (Populus tremula L.). Auxin occurred in the roots from May to October but not in November. The highest auxin level was found during the period of shoot growth. During this period the capacity of root segments to form suckers was low. Auxin level decreased in isolated root segments during the first 24 hours after excision and was low during the period of sucker induction. The relation of endogenous auxin level to control of sucker formation is discussed. The experiments do not exclude the possibility that the auxin effect is mediated through inhibitors.     

Ethylene, seed germination, and epinasty

Ethylene activity in lettuce seed (Lactuca satina) germination and tomato (Lycopersicon esculentum) petiole epinasty has been characterized by using heat to inhibit ethylene synthesis. This procedure enabled a separation of the production of ethylene from the effect of ethylene. Ethylene was required in tomato petioles to produce the epinastic response and auxin was found to be active in producing epinasty through a stimulation of ethylene synthesis with the resulting ethylene being responsible for the epinasty. In the same manner, it was shown that gibberellic acid stimulated ethylene synthesis in lettuce seeds. The ethylene produced then in turn stimulated the seeds to germinate. It was hypothesized that ethylene was the intermediate which caused epinasty or seed germination. Auxin and gibberellin primarily induced their response by stimulating ethylene production.     

Auxin Transport in Secondary Tissues1

Auxin transport was investigated in excised stem segments ofNicotiana tabacum L. by the agar block technique using [1-¹⁴C]indol-3yl-acetic acid (IAA). The ability of the stems to transportauxin basipetally increased as secondary development proceeded;by contrast the ability of the pith to transport auxin declinedwith age. By separation of the stem tissues it was shown thatthe great majority of auxin transport took place in cells associatedwith the internal phloem and in cells close to the cambium;in both cases similar velocities of transport were found (c.5.0 mm h^–1 at 22°C). The effects of osmotic gradientson auxin transport through the internal phloem were investigated.IAA was found by chromatography to account for practically allthe radioactivity in receiver blocks and other extracts of stemsegments. The significance of these results is discussed.     

Regulation of ethylene production by phosphate in Penicillium digitatum

Inorganic phosphate regulated ethylene production in shake culturesof Penicillium digitatum. Decreasing the phosphate level ofthe medium from 100 to 0.01 mM markedly increased, about 100-fold,the rate of ethylene production, in 96 hr, which was confinedentirely to the fungal mycelium. Exogenous addition of between0.01 to 100 mM phosphate, to high ethylene producing, low-phosphatecultures strongly inhibited their ethylene production and increasedthe ATP content of the mycelium. Phosphate also inhibited ethyleneproduction in apple slices. Addition of calcium ions to theincubation medium stimulated the production of ethylene in appleslices, subhook epicotyl segments of pea and shake culturesof P. digitatum. We suggest that this stimulatory effect wascaused by the reduction of inhibitory levels of phosphate, whichcomplexed with calcium. Thus, phosphate in conjunction withcalcium may play an important role in regulating ethylene productionnot only in P. digitatum but also in higher plants. ¹ On leave from the Agricultural Research Organization, TheVolcani Center, Israel. ² On leave from the M.S. University of Baroda, India. (Received September 7, 1977; )     

Growth and cell wall changes in rice coleoptiles growing under different conditions II. Auxin-induced growth in coleoptile segments

The effect of auxin on growth, mechanical properties of thecell wall and cell wall sugar composition in rice coleoptilesegments excised at different ages from seedlings growing underdifferent conditions were investigated. Auxin markedly inducedgrowth only in segments excised from coleoptiles in the fastgrowth phase with a high content of non cellulosic glucose intheir cell walls. The response to auxin decreased with coleoptileage, accompanying a decrease in the amount of the noncellulosicglucose in the cell wall, suggesting a correlation between noncellulosicglucose content and growth capacity in response to auxin. Goodcorrelation among auxin-induced growth, auxin-induced decreasein the T_o value and auxin-induced decrease in the noncellulosicglucose content of the cell wall also was found. ¹ Present address: Departamento Fisiologia Vegetal, Facultadde Ciencias, Universidad de Salamanca, Salamanca, Spain. (Received May 21, 1979; )     

Auxin Stimulates Cl-Uptake by Oat Coleoptiles

The effects of auxin on net ion fluxes near parenchyma of oatcoleoptiles were studied using the non-invasive MIFE systemto measure specific ion fluxes using ion selective microelectrodes.Application of 10 µM1-naphthaleneacetic acid (NAA) for3 h caused doubling of coleoptile segment growth, without changingthe pH of the unbuffered bathing solution from pH 5.4 duringthat time. Short term experiments revealed that auxin led toan immediate three-fold increase of chloride influx to 1200nmol m^-2s^-1, maintained for at least 1 h. In the first minutesafter auxin application, proton fluxes were small (-25 nmolm^-2s^-1, an efflux) and tended to decrease, whereas potassiumand calcium fluxes changed little, fluctuating from -100 to0 nmol m^-2s^-1and from -15 to 0 nmol m^-2s^-1, respectively. Itis suggested that one target of auxin action in plant cellsis the plasma membrane chloride transport system mediating increasedchloride uptake.Copyright 1998 Annals of Botany Company Auxin, chloride transport, ion flux,Avena sativaL., oat.     

Rapidly Induced Wound Ethylene from Excised Segments of Etiolated Pisum sativum L., cv. Alaska: I. Characterization of the Response

A rapidly induced, transitory increase in the rate of ethylene synthesis occurred in wounded tissue excised from actively growing regions of etiolated barley, cucumber, maize, oat, pea, tomato, and wheat seedlings. Cutting intact stems or excising 9-mm segments of tissue from near the apex of 7-day-old etiolated Pisum sativum L., cv. Alaska seedlings induced a remarkably consistent pattern of ethylene production. At 25 C, wound-induced ethylene production by segments excised 9 mm below the apical hook increased linearly after a lag of 26 minutes from 2.7 nanoliters per g per hour to the first maxium of 11.3 nanoliters per g per hour at 56 minutes. The rate of production then decreased to a minimum at 90 minutes, increased to a lower second maximum at 131 minutes, and subsequently declined over a period of about 100 minutes to about 4 nanoliters per g per hour. Removal of endogenous ethylene, before the wound response commenced, had no effect on the kinetics of ethylene production. Tissue containing large amounts of dissolved ethylene released it as an exponential decay with no lag period. Rapidly induced wound ethylene is synthesized by the tissue and is not merely the result of facilitated diffusion of ethylene already present in the tissue through the newly exposed cut surfaces. Previously wounded apical sections did not exhibit a second response when rewounded. No significant correlation was found between wound-induced ethylene synthesis and either CO₂ or ethane production.     

Rapid Production of Auxin-induced Ethylene

The time course of auxin-induced ethylene production was determined in mesocotyl segments of etiolated sorghum (Sorghum bicolor L. Moench) seedlings. The latent period between addition of auxin and a detectable rise in ethylene release was 15 to 20 minutes in four different genotypes. This may indicate that the initial effect of auxin on ethylene production is too rapid to involve synthesis of an ethylene-producing enzyme. The technique devised for these experiments involves placing tissue segments end to end in a glass tube, and it allows simultaneous determination of growth and ethylene production.     

Nucleic Acid Metabolism During Early Development of Pollinated and Auxin-induced Parthenocarpic Watermelon Fruits

Following pollination or induction of parthenocarpy by NAA¹treatment watermelon ovaries synthesize large quantities ofRNA. Hormone-treated ovaries increase their rate of RNA synthesisover untreated controls by 6 h after treatment whereas pollinatedovaries do so by 24 h. The lag period with pollinated ovarieshas been related to the growth of pollen tubes toward the ovulesand it is suggested that auxin secretion by the growing pollentubes may be the stimulus for increased RNA synthesis. Evidenceis presented that the cells in the flesh of developing ovariesbecome polyploid as they enlarge.     

THE INTERACTION OF AUXIN AND ETHYLENE IN THE MAINTENANCE OF LEAF BLADE FORM IN PHASEOLUS VULGARIS L. VAR. PINTO

Auxin treatment results in hyponastic curvature of the primary leaves of Phaseolus vulgaris L. var pinto. Ethylene production by hyponastic leaves is detected within 1 hr after treatment with IAA in concentrations at or above 1 μm. The amount of ethylene detected is proportional to the concentration of auxin applied. Untreated control leaves and leaves treated with 2,3,5-tri-iodobenzoic acid or gibberellic acid did not produce ethylene detectable by our equipment. The hyponastic curvature induced by auxin treatment can be inhibited by exogenous application of ethylene or ethylene-generating compounds, and these treatments produce epinasty in auxin-treated leaves. Treatment with inhibitors of ethylene synthesis or action, such as aminoethoxy-vinylglycine, carbon dioxide, or heat treatment, prolong hyponasty. The planar form, therefore, appears to be affected by both hyponastic auxin effect and an epinastic ethylene effect.     

Effects of Inhibitors of Protein Synthesis on Transmembrane Auxin Transport in Cucurbita pepo L. Hypocotyl Segments

Pretreatment of 2?0 mm segments of etiolated zucchini (Cucurbitapepo L.) hypocotyl with cycloheximide (CH) or 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide(MDMP) eliminated the stimulation by N-1-naphthylphthalamicacid (NPA) of net uptake of [1-¹⁴C]indol-3yl-acetic acid ([1-¹⁴C]IAA),but had relatively little effect on the net uptake of IAA inthe absence of NPA. The efflux of [1-¹⁴C]IAA from preloadedsegments was not substantially affected by inhibitor pretreatmentin the absence of NPA, but CH pretreatment significantly inhibitedthe reduction of efflux caused by NPA. Pretreatment with CHor MDMP did not affect net uptake by segments of the pH probe[2-¹⁴C]5,5-dimethyl-oxazolidine-2,4-dione ([2-¹⁴C]DMO), or thenet uptake of [¹⁴C]-labelled 3-O-methylglucose ([¹⁴C]3-0-MeGlu),suggesting that neither inhibitor affected intracellular pHor the general function of proton symporters in the plasma membrane.Both compounds reduced the incorporation of label from [³⁵S]methionineinto trichloroacetic acid (TCA)-insoluble fractions of zucchinitissue, confirming their inhibitory effect on protein synthesis. The steady-state association of [³H]IAA with microsomal vesiclesprepared from zucchini hypocotyl tissue was enhanced by theinclusion of NPA in the uptake medium. The stimulation by NPAof [³H]IAA association with microsomes was substantially reducedwhen the tissue was pretreated with CH. However, CH pretreatmentdid not affect the level of high affinity NPA binding to themembranes indicating that treatments did not result in lossof NPA receptors. It is suggested that the auxin transport site on the effluxcarrier system and the receptor site for NPA may reside on separateproteins linked by a third, rapidly turned-over, transducingprotein. Key words: Auxin carriers, auxin efflux, Cucurbita pepo, phytotropin receptors     

Inhibition of auxin-induced ethylene production by salicylic acid in mung bean hypocotyls

Salicylic acid (SA), a common plant phenolic compound, influences diverse physiological and biochemical processes in plants. To gain insight into the mode of interaction between auxin, ethylene, and SA, the effect of SA on auxininduced ethylene production in mung bean hypocotyls was investigated. Auxin markedly induced ethylene production, while SA inhibited the auxin-induced ethylene synthesis in a dose-dependent manner. At 1 mM of SA, auxininduced ethylene production decreased more than 60% in hypocotyls. Results showed that the accumulation of ACC was not affected by SA during the entire period of auxin treatment, indicating that the inhibition of auxin-induced ethylene production by SA was not due to the decrease in ACC synthase activity, the rate-limiting step for ethylene biosynthesis. By contrast, SA effectively reduced not only the basal level of ACC oxidase activity but also the wound-and ethylene-induced ACC oxidase activity, the last step of ethylene production, in a dose-dependent manner. Northern and immuno blot analyses indicate that SA does not exert any inhibitory effect on the ACC oxidase gene expression, whereas it effectively inhibits both the in vivo and in vitro ACC oxidase enzyme activity, thereby abolishing auxin-induced ethylene production in mung bean hypocotyl tissue. It appears that SA inhibits ACC oxidase enzyme activity through the reversible interaction with Fe²⁺, an essential cofactor of this enzyme. These results are consistent with the notion that ethylene production is controlled by an intimate regulatory interaction between auxin and SA in mung bean hypocotyl tissue.