Tracheary Element Differentiation Induced in Isolated Cylinders of Lettuce Pith: a Bipolar Gradient Technique

To study the influence of morphogenetic gradients on vasculardifferentiation patterns, a new technique was developed whichallows different substances to be applied at opposite ends ofa tissue block. It yielded information on the mobility of particularmorphogens and on the dependence of callus formation and trachearyelement differentiation on their presence. Application of indol-3ylacetic acid (1AA) (10 mg l^–1), zeatin (0.1 mg l^–1)and sucrose (3 per cent, w/v) in various combinations to theends of cylindrical explants of lettuce pith (Lactuca sativaL.) showed that (a) callus formation was stimulated by IAA,whereas induction of tracheary elements required both IAA andzeatin; (b) callus was confined to a few millimetres at theends of the explants, and tracheary elements occurred mainlywithin the callus; (c) sucrose or its metabolic products diffusedthe 10 mm length of the explants, while IAA and zeatin wereeffective only close to the application site; and (d) some callusand tracheary elements formed when no sucrose was applied, butboth increased with sucrose application, though inhibition oftracheary elements formation occurred with high sucrose concentrations. differentiation, pith explant, tissue culture, xylogenesis, indol-3yl acetic acid, sucrose, zeatin, lettuce, Lactuca sativa     

Effect of some Metabolic Inhibitors on Rooting Cuttings of Phaseolus mungo under Varying Light Conditions and its Relationship with Auxin and Nutrition

Water and 1 mg1^–1 each of IAA and IBA completely inhibitedon the cuttings of Phaseolus mungo obtained from the seedlingsraised under far-red light but rooting took place in the darkand under white and red lights. Sucrose, however, caused rootingunder far-red light and its effectiveness increased with theaddition of IAA and IBA to the sucrose medium IBA being moreeffective. Culturing in 1 and 5 mg 1^–1 each of FudR, actinomycin-D,cycloheximide and chloramphenicol after pre-treatment with water,IAA, IBA, sucrose and IAA/IBA + sucrose inhibited rooting. Theeffect increased with the concentration of each inhibitor. Incontrast to this, the culturing in water, IAA, IBA, sucroseand IAA/IBA + sucrose after pre-treatment with these metabolicinhibitors produced varying effects. While the inhibition persistedin water, IAA and IBA and even in 5 mg 1^–1 of each inhibitorthat in sucrose stimulated rooting and the effect increasedwith the addition of IAA and IBA to sucrose, the effect of IBA+ sucrose being more pronounced. This stimulation was irrespectiveof the inhibitor and light condition except that 1 mg 1^–1actinomycin-D inhibited rooting in the dark and under far-redlight.     

Germination of Spirodela polyrhiza turions: the role of culture conditions during turion development

The rapidly germinating "old" turions of Spirodela polyrhizawere shown to derive mainly from the slowly germinating "young"turions. This modification to "old" turions could occur evenin isolated "young" turions, and was accelerated by sucrose.It is suggested that this modification is a form of turion senescenceand that turion initiation and maturation are strongly influencedby exogenous carbon and nitrogen sources. (Received November 19, 1979; )     

The Relationship between the Growth of the Primary Leaf and of the Coleoptile in Seedlings of Avena and Triticum

Partial inhibition of extension growth of the primary leaf occurswhen whole Triticum seedlings are immersed in aerated solutionsof IAA but is replaced by growth promotion when sucrose is addedto the external solution. In seedlings in which the coleoptilehas been excised, IAA increases the growth of the leaf bothwith and without additional sucrose. Inhibition of the leaf by moderate concentrations of IAA nolonger occurs when the seedling is detached from the endosperm.Sucrose added to the external solution raised the percentageelongation of the coleoptile almost to the level of that attainedin intact seedlings without additional carbohydrate. It alsoenabled the leaf to show a positive growth response with IAA. The results indicate that in intact seedlings treated with IAAthe growth of the primary leaf is markedly diminished owingto diversion of carbohydrate to the coleoptile if the growthof the latter is promoted as a result of the treatment. Whenthe competition of the coleoptile for carbohydrate is diminishedor eliminated, acceleration of the growth of the primary leafby IAA becomes apparent. In addition to the endogenous rhythm, with a period close to24 hours, induced in the growth-rate of the coleoptile whenseedlings of Avena are transferred from red light to darkness,a similar rhythm, with a slightly longer period, is inducedin the growth-rate of the primary leaf. This rhythm persistsin elongating leaves so long as they remain within the coleoptile.It can be recorded for at least 100 hours in deseeded seedlings. When intact seedlings of Avena are immersed for one hour inrelatively high concentrations of IAA and then transferred todistilled water for 18 hours, the elongation of the coleoptileis greater and the inhibition of the leaf is less than whenthey are transferred to humid air. Sections of the leaf of Triticum showed a slight increase inelongation in concentrations of IAA up to 5 mg./l., but no evidencewas obtained that sections of leaf and coleoptile exert any.influenceon each other's elongation when floated together on solutionsof IAA.     

Stimulation of Nitrate Reductase by Light and Ammonium in Spirodela oligorrhiza

Light-enhanced nitrate reductase (NR) activity was 8 times greaterthan the dark control. Exogenous application of sucrose, glucoseand fructose increased the induction of NR in the light as wellas in the dark, whereas glycolate had no effect. DCMU [3-(3,4-dichlorophenyl)-1, 1-dimethyl urea] completely inhibited thedevelopment of NR in light. Sucrose, when added with DCMU, reversedthis inhibitory effect NR in vivo was more stable in light thanin darkness, the half-lives being 9.6 h and 6.4 h, respectively.The addition of sucrose did not change the half-life of NR ineither light or darkness. Ammonium, the end product of the inorganicnitrogen assimilatory pathway, stimulated the NR activity whereasamino acids decreased it. Key words: Spirodela oligorrhiza, nitrate reductase, ammonium, light     

Effect of Ethylene on Sucrose Uptake in Root Discs of Sugar Beet

Exogenously-added ethylene stimulated active sucrose uptakein root discs of sugar beet (Beta vulgaris L.) in a log dose-linearresponse manner. The ethylene precursor, 1-aminocyclopropane-1-carboxylicacid (ACC) stimulated both endogenous ethylene production andsucrose uptake. Conversely, an inhibitor of ACC synthesis, aminoethoxyvinylglycine(AVG) inhibited both endogenous ethylene production and sucroseuptake. Exogenously-added ethylene can overcome the AVG effecton sucrose uptake. Root tissue from freshly-harvested sugarbeet plants contain gas-phase ethylene levels slightly belowthat required to stimulate active sucrose uptake. No differenceswere found in gas-phase ethylene levels in the root tissue ofsugar beet cultivars having different concentrations of sucrose.The root tissue has an inherent capacity to synthesize ACC andethylene at high rates. Like ethylene, propylene can stimulate active sucrose uptakein beet root discs, but it is not detected in the gas phaseof the tissue. Acetylene, propane, and ethane had no effecton sucrose uptake. Exogenously-added IAA and ABA each make ethylenesensitivetissue insensitive to ethylene stimulation of sucrose uptake.Other plant hormones have no apparent effect on the ethyleneresponse. The role that ethylene may play on sucrose uptakein root tissue of sugar beet is discussed. (Received February 12, 1986; Accepted April 22, 1986)     

The Influence of Mannitol and Auxin on Growth of Cell Walls in Avena Coleptiles

Studies were made on the structure of cell walls in Avena coleoptilesegments incubated in sucrose and buffer without IAA (indoleaceticacid), with 8 p.p.m. IAA, and with 8 p.p.m. IAA+o·2 or0·3 M mannitol respectively. Examination in electronand polarizing microscopes revealed no difference in the structureof walls from segments grown in IAA and in IAA+mannitol. Measurementsof the weight of wall material showed that in the presence ofmannitol, wall deposition was inhibited. Measurements on theuptake of oxygen by segments showed that mannitol eliminateda respiratory stimulus produced by IAA. It is suggested thatnew wall material is only deposited on cell walls which arebeing thinned by elongation, and that the respiratory stimulusproduced by IAA and eliminated by mannitol may be in part amanifestation of metabolism involved in the synthesis of newwall material. Wall extension does not appear to be dependenton deposition of new material.     

Growth responses of sunflower hypocotyl disks inoculated with Escherichia coli and Agrobacterium tumefaciens I. Morphological observations

Disks of sunflower hypocotyls 1 mm thick grown in light anddarkness on agar containing mineral salts and sucrose to whichIAA was added in varying concentrations, were inoculated withE. coli, A. tumefaciens or sterile synthetic medium. Light-growndisks inoculated with E. coli proliferated from the lower surfaceand formed numerous long roots but dark-grown disks were usuallyinhibited in comparison with uninfected disks. Inoculation withA. tumefaciens induced proliferation mainly from the upper surfaceand a few short roots were formed. Uninfected disks grown with0.01 ppm IAA proliferated in a manner similar to that of E.coli-infected light-grown disks on simple medium but in thedark similar treatments produced quite different morphologicaleffects. The form of proliferation induced by E. coli underall conditions of growth could not be equated with that inducedby A. tumefaciens or by different concentrations of IAA. (Received December 5, 1967; )     

Peroxidase in Tobacco Abscission Zone Tissue: VII. ULTRAVIOLET ABSORPTION SPECTRA OF ETHYLENE-TREATED TOBACCO FLOWER PEDICEL TISSUE

Peroxidase-containing enzyme supernatant preparations of tobaccoabscission zone flower pedicel tissue sections of ethylene-treatedand untreated Nicotiana tabacum L. plants were assayed for u.v.absorption spectra with and without the addition of IAA. Theu.v. spectral absorption of peroxidase-catalysed IAA oxidationindicated that there is not an appreciable difference in absorbancepeaks in the ethylene-treated plants compared with untreatedcontrols, in the presence of added IAA; however, the ethylene-treatedtissues alone show different absorption peak values in the 1,2, 3, 4, 4.5, and 5 h samples. These results are discussed inrelation to ethylene-induced peroxidase activity in tobaccoflower pedicel abscission zone tissue homogenates.     

Auxin-promoted Transport of Metabolites in Stems of Phaseolus vulgaris L: EFFECTS AT THE SITE OF HORMONE APPLICATION

The hypothesis that indol-3yl-acetic acid (IAA) stimulates solutemobilization in stems by increasing metabolite utilization (i.e.sink strength) and/or sieve tube unloading mechanisms was investigatedusing seedlings of Phaseolus vulgaris L. Sink strength was assessedby obtaining estimates of rates of growth, ¹⁴C incorporationinto protein, sucrose metabolism, and sucrose uptake. The dataobtained suggest that, whilst enhancing assimilate mobilization,IAA had no short term effect on the sink strength of the treatedstumps. In several experiments involving long distance transportof ¹⁴C-assimilates, the magnitude of auxin-promoted transportwas found to be insensitive to potential changes in sink strength.Furthermore, ¹⁴C transport profiles demonstrated that the siteof hormone action was not confined to its point of application.     

The Effect of High Concentrations of Auxin on the Extension of Avena Coleoptile Sections

The response of Avena coleoptile sections to high concentrationsof auxin has been determined in the absence of all additivesexcept sucrose. In most experiments the growth-time curves with75 p.p.m. IAA showed two linear phases. In the first phase,which lasted for only 2–4 hours, extension was as rapidwith 75 p.p.m. IAA as with 5 p.p.m. IAA. This rapid initialexpansion phase was then succeeded by a second phase which persistedfor at least 20 hours. During this second linear phase the growth-ratewith 75 p.p.m. IAA was lower than with an auxin concentrationof 5 p.p.m. In some experiments the first phase was absent andonly the second phase was present. The response of sections to high concentrations of auxin wasnot influenced by the presence of buffers or absorbable cations.Omission of sucrose or the presence of moderate amounts of ethanolcaused the resulting growth curves to be non-linear. The rate of uptake of auxin into the tissues was dependent onthe auxin concentration and was constant for at least 24 hours.     

Effect of Temperature on the Nitrogenase Activity of Intact and Detached Nodules in Lotus and Stylosanthes

Temperature and plant age influenced the nitrogenase activityof Lotus and Stylosanthes nodules. Time course studies usingnodulated plants in closed vials showed a decline in activityafter 48 h; regassing with 10% (v/v) acetylene in air partiallyrestored the activity. Transfer of plants from 15, 20, 25, and30 ?C to 40 ?C immediately stopped activity; this was completelyrestored within 1 h after return to original temperatures. Detached nodules cultured on nitrogen-free agar medium exhibiteda sucrose concentration (2–8%, w/v)-dependent nitrogenaseactivity at each temperature. With 6% sucrose prolonged activity,up to 96 h, was obtained. Decline in nitrogenase activity indetached nodules was due partly to exposure to air during excisionand transfer to the medium.     

Auxin-Promoted Transport of Metabolites in Stems of Phaseolus vulgaris L.: EFFECTS REMOTE FROM THE SITE OF HORMONE APPLICATION

Phloem transport in stems of Phaseolus vulgaris was found tobe sensitive to treatment with the auxin transport inhibitor,2,3,5-triidobenzoic acid (TIBA). The response was dependenton the concentration of TIBA applied. A concentration of TIBA(0?5% in lanolin) which did not interfere with normal phloemtransport proved inhibitory to both basipetal transport of IAAand the acropetal component of IAA-promoted metabolite transport.In contrast, both acropetal IAA transport and basipetal IAA-promotedmetabolite transport were unaffected by TIBA treatment. Theinhibitory effect of TIBA on acropetal IAA-promoted transportwas overcome by providing IAA below the point of TIBA application.Both acropetal and basipetal IAA-promoted transport in stemsegments were unaccompanied by any corresponding changes inthe accumulation of [¹⁴C]sucrose by the segments.     

Phytochrome Control of Turion Formation in Spirodela polyrhiza L. Schleiden

Turion yield in Spirodela polyrhiza, strain SJ, is increasedby increasing the daily light period. This effect is more pronouncedin autotrophic than in mixotrophic conditions. Night-break irradiation(15 mins) increased turion yield by 150 % under the conditionsof an 8-h daily light period. Besides the effect of night-breakirradiation, end-of-day far-red irradiation decreased turionyield with increasing photoperiod, whereas end-of-day red irradiationwas without any effect. This demonstrates the promoting effectof the P_fr form of phytochrome on formation of light-grown turions. Formation of dark-grown turions was increased by about 240%by a single red light pulse and was reversed by an immediatelyapplied far-red light pulse. Consequently, under heterotrophicconditions phytochrome modulates the turion formation process. Spirodela polyrhiza L. Schleiden, duckweed, Lemnaceae, photomorphogenesis, phytochrome, turion     

Effects of Auxin and Phenobarbital on Morphogenesis and Production of Digitoxin in Digitalis Callus

Pieces of callus obtained from seedlings of Digitalis purpureawere grown on solid Murashige-Skoog's medium supplemented with1 mg liter^–1 BA and 0.1 mg liter^–1 IAA or NAA, withor without phenobarbital (40 mg liter^–1). The replacementof the natural auxin IAA by the synthetic auxin NAA increasedcallus growth and inhibited organogenesis, whereas the additionof phenobarbital had the opposite effect. Morphometric measurementsrevealed a high ratio of vacuole to cytoplasm (v/v) in calluscells. This ratio was affected by the different treatments inthe same way as the fresh weight. The activity of mitochondrialcytochrome P450scc (the enzyme that provides the precursor,pregnenolone, for the biosynthesis of cardenolide in foxgloveplants) was detected in the relevant fraction of callus grownunder all experimental conditions, and its activity was increasedby the addition of phenobarbital. The different treatments testedincreased the cardenolide content and quantifiable amounts ofdigitoxin were detected in all callus tissues. It is of specialinterest that phenobarbital added to the culture medium increasedthe accumulation of digitoxin. The mechanism affecting the developmentand production of cardenolide in callus tissues of D. purpureaby phenobarbital and the replacement of IAA by NAA is discussed. (Received July 18, 1994; Accepted December 14, 1994)     

Short-term Effects of Some Chemicals on Cambial Activity

Aqueous solutions of indol-3yl-acetic acid (IAA), 1-naphthyl-aceticacid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), gibberellicacid (GA), 6-furfuryl-aminopurine (FAP), myo-inositol, and sucrosewere applied singly and in mixtures to the apical ends of disbuddedstem segments of willow. After 4 weeks all substances had hadsome effect on differentiation of xylem from cambial derivatives.The production of potential xylem cells, as well as their differentiationwere most markedly enhanced when IAA, GA, and FAP were appliedtogether, although the response was further augmented by additionof inositol or sucrose. The action of the substances when appliedas mixtures was often synergistic. This means that it is difficultto assess the role of different chemicals in xylem productionby extrapolation from experiments involving the applicationof single substances.     

Levels of IAA, Cytokinins, ABA and Ethylene in Rice Plants as Affected by a Gibberellin Biosynthesis Inhibitor, Uniconazole-P.

Effects of uniconazole-P, a triazole-type growth retardant,on endogenous levels of IAA, cytokinins, ABA and ethylene inrice seedlings were investigated. Endogenous levels of IAA andABA were similar between control and uniconazole-P-treated riceshoots. Evolution of ethylene was promoted slightly, being 1.8times greater under 0.3 ppm uniconazole-P treatment than thatof control. The most obvious effect was the increase of trans-Zand trans-RZ in shoots. Shoots treated with uniconazole-P (10mg/m² nursery box) contained 3.4 times and 3 times more trans-Zand trans-RZ than control, respectively. No significant differencesof cytokinin levels were recognized in roots except for cis-RZ.The increase of ethylene and active forms of cytokinins, andthe decrease of gibberellin in the shoots may be the basis forphysiological phenomena caused by uniconazole-P, namely thepromotion of flowering in woody plants and the enhancement offemaleness in cucumber. (Received September 9, 1987; Accepted October 20, 1987)     

Osmoregulation in Rice Coleoptile Elongation as Promoted by Cooperation between IAA and Ethylene

IAA-induced elongation of rice (Oryza sativa L. cv. Sasanishiki)coleoptiles is regulated by cooperation between IAA and ethyleneproduced in response to IAA. However, the presence of some solutes,such as K^$, Na^$, Rb^$, glucose and sucrose, in the incubationmedia was found to be indispensable for this cooperation. Withoutthose solutes, the IAA-induced elongation was not sustainedover a long time period. IAA caused increases in both the osmoticpotentials of the coleoptile cells and the extensibility oftheir cell wall. In epidermal cells of IAA-treated coleoptiles,the osmotic potential increased from –0.87 to –0.62MPa during a 4-h incubation with 1 mM KCl. Moreover, IAA promotedthe uptake of K^$ or Na^$ from the media into the coleoptiles.However, these effects of IAA were partially prevented by aminoethoxyvinylglycine(AVG), and all the AVG effects were completely nullified byethylene applied simultaneously and exogenously. Both IAA andethylene did not affect the wall yield stress. These resultssuggested that the long-term elongation induced by IAA in ricecoleoptile segments results from inhibiting increases in osmoticpotentials of their cells. The maintenance by IAA of low osmoticpotentials may be partly due to the promotive action of ethyleneproduced in response to IAA on the solute uptake from the media. (Received July 6, 1983; Accepted February 15, 1984)     

Effects of Osmotic Stress, Ionic Stress and IAA on the Cell-Membrane Resistance of Vigna Hypocotyls

The cell-membrane resistance (R_m) of Vigna hypocotyls was examined,and the effects of osmotic stress, ionic stress and IAA on R_mwere investigated. R_m decreased by 64 to 77% under osmotic stressin the presence of absorbable solutes (40 mM sorbitol, 15 mMKC1, 30 mM sucrose; or 40 mM sorbitol, 15 mM KC1, 30 mM sucroseplus 10^–4 M IAA) or under ionic stress (50 mM NaCl or50 mM KC1). R_m was not changed by perfusion with 10^–4M IAA. Therefore, the hyper-polarizations of the membrane potentialobserved in both cases should be ascribed totally to the activationof the electrogenic proton pump. Although R_m showed an increaseof 1.6 fold when the hypocotyls were subjected to osmotic stress(100 mM sorbitol or 100 mM sorbitol plus 10^–4 M IAA),83.6% or 92.4% of the hyperpolarization of the membrane potential(V_px was also the result of the activation of the pump. Theresults, calculated on the basis of the current source model,support the viewpoint that the hyperpolarization of the cellmembrane potential of Vigna hypocotyls under osmotic stress,ionic stress or in the presence of IAA is an expression of theactivation of the proton pump, and is not caused by an increasein R_m. ¹ Present address: Researchers and Planners of Natural Environment, Yotsugi Bldg. (2F), 1-5-4 Horinouchi, Suginami-Ku, Tokyo,166 Japan ² Present address: Graduate School of Integrated Science, YokohamaCity University, 22-2 Seto, Kanazawa-Ku, Yokohama, 236 Japan (Received February 14, 1991; Accepted July 24, 1991)     

The Effect of Mutations in the T-DNA Encoded Auxin Pathway on the Endogenous Phytohormone Content in Cloned Nicotiana tabacum Crown Gall Tissues

We analyzed the endogenous auxin and cytokinin levels of clonedNicotiana tabacum SR 1-lines induced either by the wild-typeAgrobacterium tumefaciens C58 strain or by mutants affectedin the T-DNA-encoded IAA biosynthesis pathway. The wild-typeSR1-C58 line contained up to 20 times more IAA than a nontransformedSRI-callus line. The mutant lines affected in gene 1 (iaaM^–)or gene 2 (iaaH^–) contained intermediate levels of IAA. Analysis of the endogenous levels of indole-3-acetamide (IAM)in the nontransformed SR 1 callus line, the wild-type SR1-C58and the two mutant lines confirmed the T-DNA-induced IAA biosynthesispathway in the transformed tumor cells. Supplementing auxinto the mutant lines resulted in complete suppression of theshoot-forming ability, but no changes in the endogenous IAAlevels. There was no marked difference in the cytokinin level betweenthe nontransformed callus line and the wild type tumor line.The two mutant lines, however, showed a 20- to 30-fold highercytokinin level which was not affected by the addition of NAA.The T-DNA encoded hormone biosynthetic pathways are discussedin relation to pathways of the host plant. (Received July 29, 1986; Accepted February 14, 1987)