Time sequence of the effect of ethylene on transport, uptake and decarboxylation of auxin

The effect of ethylene on the uptake, decarboxylation and basipetaltransport of IAA-1-¹⁴C, IAA-2-¹⁴C and NAA-1-¹⁴C in cotton stemsections (Gossypium hirsutum L., var. Stoneville 213) was studied.A reduction in the capacity of cotton stem sections to transportauxin basipetally appears in sections excised from plants exposedto ethylene for only 3 hr and increases with fumigation time. In addition to reducing transport, increasing ethylene pretreatmentperiods from 3 to 15 hr also progressively reduced the uptakeof ¹⁴C and increased the release of ¹⁴C as ¹⁴CO₂ from IAA-1-¹⁴C.The effect of ethylene on the decarboxylation of IAA-1-¹⁴C wassignificant when expressed as either the cpm of ¹⁴C releasedper hr per mg dry weight or the cpm released per hr per mm²in contact with the IAA donor. Comparative experiments usingIAA-1-¹⁴C and IAA-2-¹⁴C demonstrated that the effect of ethyleneon the decarboxylation of IAA was primarily a cut surface effectwhich apparently contributes to the reduction of IAA uptakeby ethylene. Although ethylene significantly reduced the transport of NAA-1-¹⁴C,uptake was significantly increased rather than decreased aswith IAA-1-¹⁴C while decarboxylation was unaffected. Ethylene pretreatment caused no significant changes in the dryweight or the cross-sectional area of the absorbing surfaceof the transport tissue. ¹A contribution of the Texas Agricultural Experiment Station.Supported in part by Grant GB-5640, National Science Foundationand grants from the Cotton Producers Institute and the NationalCotton Council of America. ²Present address: Central Research Department, E. I. Du PontDe Nemours and Company, Wilmington, Delaware 19898, U. S. A. (Received May 29, 1969; )     

Effect of kinetin on auxin uptake and distribution in normal and tumor-prone Nicotiana shoots

Stem segments of non-tumorous Nicotiana glauca and N. langsdorffiiplants and of their tumor-producing amphidiploid F₁ hybrid weretreated with 6-furfurylaminopurine (kinetin) prior to transporttests with applied labeled indoleacetic acid (IAA-2-¹⁴C). Kinetin-treatmentsincreased the uptake of IAA in non-tumorous shoots; the IAAuptake by N. langsdorffii segments was increased up to 3-fold.The auxin uptake in stem-segments of the tumor-forming hybrid,however, could not be increased significantly by kinetin. Theeven distribution of IAA-¹⁴C in segments of normal and tumorproneNicotiana shoots is stimulated by kinetin. Data are discussedin conjunction with previous results on auxin transport andtumorformation in Nicotiana. (Received August 8, 1972; )     

Kinetin Transport to Axillary Buds of Dwarf Pea (Pisum sativum L.)

Decapitation resulted in the transport of significant amountsof ¹⁴C to the axillary buds from either point of application,but pretreatment of the cut internode surface of decapitatedplants with IAA (alone or in combination with unlabelled kinetin)inhibited the transport of label to the axillary buds and resultedin its accumulation in the IAA-treated region of the stem. Inintact plants to which labelled kinetin was applied to the apicalbud there was little movement of ¹⁴C beyond the internode subtendingthis bud; when labelled kinetin was applied to the roots ofintact plants, ¹⁴C accumulated in the stem and apical bud butwas not transported to the axillary buds. A considerable proportionof the applied radioactivity became incorporated into ethanol-insoluble/NaOH-solublecompounds in the apical bud of intact plants, in internodestreated with IAA, and in axillary buds released from dominanceby removal of the apical bud. The results are discussed in relation to the possible role ofhormone-directed transport of cytokinins m the regulation ofaxillary bud growth.     

Dioxindole-3-Acetic Acid Conjugates Formation from Indole-3-Acetylaspartic Acid in Vicia Seedlings

When indole-3-acetic acid (IAA) is applied to the cotyledonsof broad bean seedlings (Vicia faba L. cv Chukyo), the majormetabolites found in the roots are 3-(O-ß-glucosyl)-2-indoIone-3-acetylaspartic acid (Glc-DIA-Asp) and 3-hydroxy-2-indolone-3-acetylasparticacid (DIA-Asp). In this report, the metabolic pathway from IAAto the two dioxindole-3-acetic acid (DIA) conjugates was investigatedby using [¹⁴C]IAA, [¹⁴C]DIA, [¹⁴C]indole-3-acetylaspartic acid(IAA-Asp), and [¹⁴C]IAA-[³H]Asp. The precursor of DIA-Asp wasfound to be IAA-Asp but not DIA. Incorporation of the doublelabeled IAA-Asp into the DIA conjugates demonstrated that hydrolysisof IAA-Asp was not involved in the formation of the DIA conjugates.DIA-Asp was further metabolized to Glc-DIA-Asp in the cotyledons,while formation of Glc-DIA-Asp in the roots was very low. Glc-DIA-Aspformed in the cotyledons was transported to the roots. (Received April 21, 1986; Accepted September 10, 1986)     

Effect of malformin on adventitious root formation and metabolism of indoleacetic acid-2-14C by Phaseolus vulgaris

Malformin inhibited rooting on cuttings of Phaseolus vulgaris.IAA antagonized malformin-induced inhibition of rooting, butmalformin inhibited IAA-induced swelling on the base of thecuttings. It was suggested that IAA-induced swelling was mediatedby ethylene. Malformin did not inhibit transport of root-promotingsubstances from upper portions of the cuttings or polar transportof IAA-2-¹⁴C, nor did it alter the melting point of DNA or thebinding of DNA to histone. Although malformin appeared to alterthe metabolism of IAA-2-¹⁴C, the effect may have been the resultof a marked and selective stimulation of efflux of IAA-2-¹⁴Cmetabolites by malformin. Efflux of IAA or its metabolites maycontribute toward inhibition of rooting by malformin. ¹ Journal Paper No. 4688 of the Purdue Agricultural ExperimentStation. Supported in part by grant GB-7158 from the NationalScience Foundation. ² Present address: Botanisches Institut der Technischen UniversitätBraunschweig, 3300 Braunschweig, Humboldtstraße 1. (Received March 9, 1972; )     

In vitro Propagation of Red Cabbage (Brassica oleracea L. var. capitata)

By manipulation of various growth regulators and physical conditions,plants have been regenerated from excised roots, stem segments,cotyledons, leaves, and callus cultures of red cabbage (Brassicaoleracea var. capitata) grown under in vitro conditions. Shootbuds were induced on isolated root segments (1 cm long) culturedon Murashige and Skoog's medium and the frequency of bud formationwas greatly enhanced by the addition of kinetin (0.5 part 10^–6).Callus obtained from the seeds, cotyledons, and hypocotyl segmentscultured on a medium fortified with 2,4-D (1 part 10^–6),kinetin (0.1 part 10^–6), and coconut milk (10%, v/v) hasbeen repeatedly subcultured. The callus is slow growing, andon transference to a kinetin (2 parts 10^–6) and IAA (2parts 10^–6) medium underwent morphogenesis to give riseto plants. The significance of the propagation of red cabbageby in vitro culture is pointed out.     

Transport and Metabolism of Kinetin-8-14C in Zea mays L. Roots

The movement of kinetin has been investigated in roots of Zeaseedlings, grown in the dark, using kinetin-8-¹⁴C. Segments (10 and 11 mm) with and without an apex were used andthe following results obtained: The transport of kinetin is polarized basipetally. Kinetin-8-¹⁴C is transported in roots without vascular tissue,but the amount transported is always lower than in roots withvascular tissue. Thin-layer chromatography showed that the molecule of kinetinis metabolized in the root segments, adenine being one of theproducts.     

Transport of shoot- and cotyledon-applied indole-3-acetic acid to Vicia faba root

To clarify the participation of indole-3-acetic acid (IAA) originatingfrom the shoot in root growth regulation and the mechanism ofIAA translocation from shoot to root, the movement of ¹⁴C-IAAwhich was applied to the epicotyl or the cotyledon of Viciafaba seedlings was investigated. The radioactivity of IAA appliedto the cotyledon moved faster to the root tip than that appliedto the epicotyl. On the basis of the effect of 2,3,5-triiodobenzoic acid on IAAmovement, a comparison with ¹⁴C-glucose movement and autoradiographicexamination, the nature of IAA movement was concluded to bepolar transport from the epicotyl to the basal part of the roots,while IAA movement from the epicotyl to the cotyledon, fromthe basal part of roots to the apical part, and from the cotyledonto the epicotyl and to the root took place in the phloem. Theradioactivity from ¹⁴C-IAA applied to the cotyledon accumulatedin lateral root primordia and vascular bundles. These factssuggest that IAA produced in cotyledons may participate in theregulation of Vicia root development. (Received December 21, 1979; )     

Pathways of auxin transport in the intact pea seedling (Pisum sativum L.)

Summary When small colonies of the pea aphid [Acyrthosiphon pisum (Harris)] were established on the stem of Meteor Dwarf Pea seedlings (Pisum sativum L.), ¹⁴C was found in the honeydew 4.5 h after applying IAA-1-¹⁴C to a fully-expanded foliage leaf. In contrast, no activity was found in the honeydew or aphids 4.5 h after the application of IAA-1-¹⁴C to the intact apical bud even though the internode upon which the aphids were feeding contained high levels of ¹⁴C. The lack of radio-activity in aphids feeding on stems to which IAA-1-¹⁴C was applied via the apical bud was found not to be influenced by the internode position or by the transport interval allowed (up to 24 h).Radioactivity derived from either foliar or apical applications of IAA-1-¹⁴C was not transported through stem tissues killed by heat treatment. Xylem function was shown not to be impared by the heat treatment employed.It was concluded that the long-distance transport of IAA from the apical bud of intact pea seedlings does not take place in the phloem sieve tubes involved in the transport of metabolites from foliage leaves, or in the non-living tissues of the xylem.     

Distribution of labeled auxin in geotropically stimulated steins of cucumber and pea

The distribution of IAA-2-¹⁴C or IAA-5-³H applied to the apexin the upper and lower (with respect to gravity) halves of geotropicallystimulated stems of cucumber and pea was examined and the resultsobtained are as follows: 1. A larger amount of IAA-2-¹⁴C or IAA-5-³H was detected inthe lower than upper half of cucumber hypocotyls with 3-hr geostimulation. 2. A larger amount of IAA-2-¹⁴C was distributed in the lowerthan upper epidermis of pea epicotyls with 1-hr geostimulation. 3. Freezing autoradiography revealed that IAA-2-¹⁴C was concentratedin the vascular bundles and epidermis of cucumber hypocotyls,the distribution being affected by geostimulation only in thelatter. 4. Application of 1% TIBA in lanolin inhibited the distributionof IAA-2-¹⁴C in the lower epidermis of pea epicotyls, causingsuppression of geotropic curvature. 5. From these results, we concluded that IAA which accumulatedin the lower epidermis of the stem upon geostimulation causedthe negative geotropic curvature of the stem. (Received October 13, 1975; )     

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     

Lipid Synthesis and Ultrastructure of the Laticifers of Etiolated Euphorbia lathyris Seedlings

In 6–14-day-old etiolated seedlings of Euphorbia lashyrisa latex triterpene synthesis of 19 µg day^–1 wasrecorded. This production was proportional to stem growth. Laticiferdistribution in the cotyledons and stem was studied. In ultra-thinsections the occurrence of many mitochondria was observed. A¹⁴C-latex triterpene synthesis was measured after ¹⁴C-glucoseand ¹⁴C-sucrose uptake by the cotyledons in which most of the¹⁴C-triterpenes were synthesized. ¹⁴C-incorporation into theselipids from [1^–14C]glucose, [6-¹⁴C]glucose and [3,4^–14points to a glycolytic catabolism of glucose prior to terpenesynthesis. The possible involvement of mitochondria in thissynthesis is discussed. Euphorbia lathyris, triterpene synthesis, laticifer, latex, mitochondria, ultrastructure     

Tiller Bud Suppression in Reproductive Plants of Lolium multiflorum Lam. Cv. Westerwoldicum

The control of tiller bud growth during reproductive developmentwas investigated in experimental plants ofLolium multiflorumLam. cv. Westerwoldicum that were reduced to a main axis havinga developing but unemerged ear, elongating stem internodes,a series of expanded leaves, slow-growing tiller buds and aroot system. Isolation of the ear by excision of its base, ordecapitation so as to remove the ear together with the upperleaves, promoted the movement of ¹⁴C-assimilates to tiller buds,decapitation being the more effective treatment. Applicationof 0.1 per cent indol–3yl-acetic acid (IAA) to cut tissuesof decapitated plants diverted ¹⁴C-assimilates to upper internodesbut did not reduce import by buds, whereas application of 1.0per cent IAA both diverted labelled assimilates to upper internodesand reduced bud import. Radioactivity from [¹⁴C] IAA appliedto the upper leaves or to the ear base was recovered from budsin very small amounts; larger amounts were recovered from budsfollowing the application of labelled IAA to an elongating internode,especially from the bud at the base of the treated internode.It is suggested that tiller bud suppression may be influencedby the movement of inhibitory levels of auxin into buds fromnearby elongating stem internodes, whose activity in turn maybe controlled by the developing inflorescence and upper leaves.     

Auxin transport in intact pea seedlings (Pisum sativum L.): The inhibition of transport by 2,3,5-triiodobenzoic acid

Summary The application of 2,3,5-triiodobenzoic acid (TIBA, 10 mg·g^-1 in lanolin) to the stem of intact pea seedlings (Pisum sativum L.) inhibited the basipetal transport of ¹⁴C from indoleacetic acid-1-¹⁴C (IAA-1-¹⁴C) applied to the apical bud, but not the transport of ¹⁴C in the phloem following the application of IAA-1-¹⁴C or sucrose-¹⁴C to mature foliage leaves. It was concluded that fundamentally different mechanisms of auxin transport operate in these two pathways.When TIBA was applied at the same time as, or 3.0 h after, the application of IAA-1-¹⁴C to the apical bud, ¹⁴C accumulated in the TIBA-treated and higher internodes; when TIBA was applied 24.0 h before the IAA-1-¹⁴C, transport in the stem above the TIBA-treated internode was considerably reduced. TIBA treatments did not consistently influence the total recovery of ¹⁴C, or the conversion of free IAA to indoleaspartic acid (IAAsp). These results are discussed in relation to the possible mechanism by which TIBA inhibits auxin transport,.Attention is drawn to the need for more detailed studies of the role of the phloem in the transport of endogenous auxin in the intact plant.Abbreviations TIBA 2,3,5-triiodobenzoic acid - IAAsp indoleaspartic acid     

The Effect of Morphactin (Methyl 2-Chloro-9-hydroxyfluorene-9-carboxylate) on Basipetal Transport of Indol-3-ylacetic Acid in Hypocotyl Sections of Phaseolus vulgaris L.

The effect of morphactin (methyl 2-chloro-9-hydroxyfluorene-9-carboxylate)on basipetal transport of auxin (Indol-3-ylacetic acid-2-¹⁴C)was studied in bean (Phaseolus vulgaris) hypocotyl with thedonor-receiver block method. Morphactin (5 x 10^–6m) reduced IAA (5 x 10^–6m) transportintensity by an average of 83 per cent and auxin transport capacityby 90 per cent, but transport velocity was not affected. Morphactin did not inhibit uptake of IAA into hypocotyl tissue,but it did prevent transfer of IAA from the tissue into receiverblocks. Chromatographic analysis of the tissue after 4 h IAA-2-¹⁴Ctransport showed that 54 per cent of the total activity wasin the form of IAA in the control and 42 per cent in the morphactintreated tissue. No difference was found in the rate of decarboxylationof IAA-1-¹⁴C between control and morphactin treated tissue sections.Nor could any difference between control and morphactin be shownin the radioactivity associated with a TCA ppt fraction. Ina study of the transportable auxin pool, morphactin decreasedthe size of the pool and increased the half-life of decay ofauxin transport from 1•22 h to 3•85 h. In a kineticanalysis of the reversal of morphactin (5 x 10^–6m) inhibitionby increasing concentration of IAA-2-14C (5 x 10^–6m to2 x 10^–5m), it was shown that IAA transport resemblesMichaelis-Menten enzyme reaction kinetics, and that inhibitionby morphactin fitted a ‘mixed type’ model. IAA hada dissociation constant of 8•5 x 10^–6m and morphactinthat of 4•3 x 10^–7m with a K_m for the transport processof 8•5 x 10^–6m.     

Effect of indoleacetic acid on the penetration of leucine and glucose through epidermal tissue of onion bulb

The penetration of leucine-(U)-¹⁴C and glucose-(U)-¹⁴C throughthe bulb epidermal tissue of Allium cepa was examined in thepresence of indoleacetic acid (IAA). Not only the uptake ofleucine-¹⁴C and glucose-¹⁴C in epidermal tissue but also theirtranscellular penetration were accelerated by IAA treatment.N-Ethylmaleimide (NEM) inhibited their uptake and transcellularpenetration, and the inhibitory effect was relieved by additionalIAA. In the presence of IAA, leucine-¹⁴C and glucose-¹⁴C weremore penetrable by adaxial than abaxial application, but inthe absence almost no difference due to application side wasobserved. IAA appears to promote permeability of the epidermaltissue only to substances applied adaxially. N,N'-Dicyclohexylcarbodiimide(DCCD) showed a little inhibitory effect on the IAA-inducedpromotion of the uptake and penetration of leucine-¹⁴C appliedadaxially. Leucine-¹⁴C and glucose-¹⁴C penetrated more easilythrough killed than fresh tissue, with little difference betweenabaxial and adaxial applications. ¹ Present address: Department of Biology, Faculty of Science,Kochi University, Kochi 780, Japan. ² Present address: Department of Medical Zoology, Medical School,Mie University, Tsu 514, Japan. (Received October 13, 1977; )     

Effects of N-1-naphthylphthalamic acid on the growth and bud formation of tobacco callus grown in vitro

The effect of N-1 -naphthylphthalamic acid (NPA), indole-3-aceticacid (IAA) and kinetin on callus growth and bud formation wasstudied mainly by a tobacco callus culture method. Callus producedfrom Nicotiana tabacum var. Wisconsin 38 was used as the testplant material. Callus growth on nutrient agar containing 2mg/liter of IAA was promoted by NPA added at a concentrationof 0.5 mg/liter with 0.4 mg/liter of kinetin or by NPA addedat 5 mg/liter in the absence of kinetin. At a high concentrationof 50 mg/liter, however, NPA inhibited growth on the mediumcontaining 2 mg/liter IAA and no kinetin. Kinetin reduced thisNPA inhibition. In the presence of 0.4 mg/liter kinetin and2 mg/liter IAA, when the concentration of NPA was 50 mg/liter,buds were initiated after calluses were grown on the test mediumfor 7 weeks in dim light, but no buds formed when NPA was omittedfrom the above medium. The control of callus growth and bud initiation is based onthe active ratio of auxin (IAA) to cytokinin (kinetin) in themedium and NPA added to the medium can promote or inhibit callusgrowth and induce bud formation. Therefore, it is proposed thatNPA can itself reduce auxin activity or enhance cytokinin activityand hence change the active ratio of the two regulators. NPAmay enhance the activity of cytokinin (here supplied as kinetin)but cannot substitute for it. ¹Present address: Department of Biology, Wisconsin State University,Oshkosh, Wisconsin 54901, U. S. A. (Received March 10, 1969; )     

Studies on soluble RNA binding indoleacetic acid in etiolated mung bean hypocotyl sections

Nucleic acid was extracted by the SLS-phenol method from Phaseolusaureus hypocotyl treated with IAA-2-¹⁴C. Radioactivity in thenucleic acid fraction was found at the positions of sRNA andrRNA on an MAK column. IAA-¹⁴C was released from the radioactivecompound(s) in the sRNA fraction, by alkaline hydrolysis, butnot by ethanol extraction, or by dialysis to 2 M NaCl, 8 M urea,and 0.1 M EDTA. When the radioactive compound at the positionof sRNA on an MAK column was further re-chromatographed on aDEAE-cellulose column and on a BD-cellulose column, it was alwayslocalized only in a settled part of the fraction of each column.From this fraction IAA-¹⁴C was released by alkaline hydrolysis.Also, IAA-¹⁴C was released from the radioactive compound insRNA fraction, by RNase digestion, but not by pronase treatment.Results of these experiments suggest the existence of some kindsof sRNA binding IAA. The genesis of this sRNA binding IAA-¹⁴Cwas observed within 30 min after the supply of IAA-¹⁴C, andthe sRNA became saturated with IAA-¹⁴C about 2 hr after thebeginning of incubation. The behavior of sRNA binding IAA, representedby sRNA binding IAA-¹⁴C, may have a role in IAA induced growthof mung bean hypocotyl sections. (Received July 6, 1971; )     

The Influence of Cotyledons in Axillary and Adventitious Shoot Production from Cotyledonary Nodes of Cucumis sativus L. (Cucumber)

Cucumber explants including at least part of the cotyledon,a short section of hypocotyl, and the apical bud, are capableof producing multiple axillary buds from the seedling apex andadventitious shoots from the hypocotyl base in a medium whichcontains 2·0 mg dm^–3 of kinetin. Removal of theapical bud triples the number of shoots produced from the apexof explants with two intact cotyledons but does not affect shootproduction from explants with some or all of their cotyledonsremoved. The area of intact cotyledon also influences morphogenesis,as explants with both cotyledons removed, failed to produceadventitious shoots from the hypocotyl base. Culture in continuousdarkness entirely prevents shoot development from the explantbase, but has little influence on shoot production from theapex. The influence of endogenous growth regulators and apicaldominance on the morphogenesis of shoots in cucumber seedlingsare discussed. Key words: Cucumber, cotyledons, apical dominance, morphogenesis, adventitious shoots, Cucumis sativus