Genetic Analysis of the Effects of Polar Auxin Transport Inhibitors on Root Growth in Arabidopsis thaliana

Polar auxin transport inhibitors, including N-1-naphthylphthalamicacid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), have variouseffects on physiological and developmental events, such as theelongation and tropism of roots and stems, in higher plants.We isolated NPA-resistant mutants of Arabidopsis thaliana, withmutations designated pir1 and pir2, that were also resistantto TIBA. The mutations specifically affected the root-elongationprocess, and they were shown ultimately to be allelic to aux1and ein2, respectively, which are known as mutations that affectresponses to phytohormones. The mechanism of action of auxintransport inhibitors was investigated with these mutants, inrelation to the effects of ethylene, auxin, and the polar transportof auxin. With respect to the inhibition of root elongationin A. thaliana, we demonstrated that (1) the background levelof ethylene intensifies the effects of auxin transport inhibitors,(2) auxin transport inhibitors might act also via an inhibitorypathway that does not involve ethylene, auxin, or the polartransport of auxin, (3) the hypothesis that the inhibitory effectof NPA on root elongation is due to high-level accumulationof auxin as a result of blockage of auxin transport is not applicableto A. thaliana, and (4) in contrast to NPA, TIBA itself hasa weak auxin-like inhibitory effect. (Received April 12, 1996; Accepted September 2, 1996)     

Exogenous Auxin Effects on Lateral Bud Outgrowth in Decapitated Shoots

In 1933 Thimann and Skoog demonstrated exogenous auxin repressionof lateral bud outgrowth in decapitated shoots ofVicia faba. This evidence has given strong support for a role of auxinin apical dominance. Most, but not all, investigators have confirmedThimann and Skoog's results. In the present study, auxin treatmentswere carried out on ten different species or plant types, manyof which were treated with auxin in different forms, media andunder different light conditions. The Thimann–Skoog experimentdid work for most species (i.e. exogenous auxin did repressbud outgrowth) including thedgt tomato mutant which is knownto be insensitive to auxin in certain responses. Toxic auxinsymptoms were observed in some but not all species. The Thimann–Skoogexperiment did not work for greenhouse-grownColeus or forArabidopsis. Light was shown to reduce apical dominance inColeus andIpomoeanil . apical dominance; lateral bud outgrowth; axillary bud; auxin; IAA; decapitation; Vicia faba ; Ipomoea nil ; Pisum sativum ; Phaseolus vulgaris ; Lycopersion exculentum ; dgt ; Coleus blumei ; Arabidopsis thaliana ; Helianthus annuus ; Thimann–Skoog     

The Relationship between Endogenous Hormone Levels in the Plant and Seasonal Aspects of Cambial Activity

The auxin levels at different heights in a ring-porous (Ulmusglabra) and a diffuse-porous (Populus trichocarpa) tree werefollowed in spring. There was shown to be a correlation betweenthe pattern of spread of cambial activity and auxin gradients.The observed continuation of cambial activity after cessationof extension growth in ring-porous species is apparently dueto the fact that the mature leaves produce auxin, whereas littleauxin is produced by the leaves of diffuse-porous species underthese conditions. The spring/summerwood transition in ring-porousspecies can be related to a fall in auxin level at the timethat shoot extension-growth ceases. There is evidence of phloemproduction after the cessation of xylem production in Ailanthusaltissima and Robinia pseudacacia, but not in Betula pubescentand Acer pseudoplatanus. It is suggested that, in Ailan-thus,phloem production continues because high endogenous gibberellinlevels are present after the auxin level has been reduced byshort-day treatment.     

Estimation of hyperauxinity during tumour formation in castor bean hypocotyls

The hyperauxinity of tumourous tissues of castor bean plants(Ricinus communis L. variety Pacific 6), infected by Agrobacteriumtumefaciens (Smith and Town) Conn, strain B6, was quantitativelyestimated. The auxin extracted was found to be indole-aceticacid (IAA), which gave a typical Ehrlich's reaction, an equalR.f. value and a characteristic indole UV spectrum which weresimilar to those of authentic IAA. The Avena coleoptile straightgrowth test revealed a higher auxin concentration in the typicalR.f. region (0.2–0.4) in the tumourous tissues than inthe control. Colorimetric estimation of the auxin concentrationin developing tumourous tissues revealed fluctuating hyperauxinitiesthroughout the test period. The possible nature of this fluctuationis discussed but it is still unexplained. (Received January 28, 1975; )     

Studies on the Geotropism of Roots: III. EFFECTS OF GEOTROPIC STIMULATION ON GROWTH-SUBSTANCE CONCENTRATIONS IN VICIA FABA ROOT TIPS

The auxins contained in 5-mm. tips of horizontal Vicia fabaroots have been compared with those in tips of vertical rootsafter cold ethanol extraction, paper-chromatographic separation,and Avena mesocotyl bioassay. At about the time curvature commencesin horizontal roots there is a marked increase in the contentof an auxin corresponding to ‘AP(ii)’ of pea roots(Rf 0.35–0.65 in isobutanol/methanol/water). There areindications that this is not due to its release from an inactivebound state but that it is either synthesized de novo or maybe converted from another auxin corresponding to ‘AP(iii)’of pea roots (Rf 0.75–1.0). The literature dealing with the auxins of geotropically stimulatedorgans is reassessed and it is concluded that, with the exceptionof the Avena coleoptile, there is very little evidence favouringa simple transport redistribution of auxin under gravity; themajority of the data favour an effect of gravity on auxin metabolism.     

Auxin Concentrations in Nodes and Internodes ofImpatiens sultani

Greater concentrations of auxin at nodes than in internodes,resulting from some nodal barrier to basipetal transport, havelong been postulated as the cause of early differentiation ofinitially isolated xylem and cambium at the nodes. However,this study, using [¹⁴C] indole-3-acetic acid (IAA) applied apicallyand gas chromatography-mass spectrometry, found that in stemsofImpatiens sultanithe IAA concentrations (per unit f. wt) atnodes were similar to those in adjacent internodes, though alittle greater at nodes if expressed per unit length of stemand a little less per unit d. wt. By contrast, in decapitatedshoots and in stem explants of dicotyledons, loss of the apicalsource of basipetally flowing auxin can result in auxin drainagewith some auxin retention in the uppermost remaining nodes.When [¹⁴C]IAA was applied apically to shoots for 4 h and stemexplants were excised, the explants had no nodal accumulationinitially whereas comparable explants incubated for 20 h revealedsignificant nodal accumulation. If decapitation leads both tonodal auxin accumulation and to adventitious abscission justabove the node, this fits the hypothesis that abscission sitesare positioned where auxin concentration decreases locally inthe apical direction. Difficulties in quantifying nodal auxindynamics are discussed, and some crude estimates of metabolicrates and locations of the auxin are presented.Copyright 1999Annals of Botany Company Abscission, auxin,Impatiens sultani, indole-3-acetic acid, node.     

The effect of auxin starvation on the growth of auxin-dependent tobacco cell culture: dynamics of auxin-binding activity and endogenous free IAA content

The growth of a cell strain derived from the stem pith of tobacco(Nicotiana tabacum L., cv. Virginia Bright Italia) was investigatedin subcultures grown at various levels of synthetic auxins.Both partial and complete auxin starvation resulted in a decreaseof the frequency of cell division. For these treatments theendogenous free indole-3-acetic acid content increased substantiallyat the commencement of the exponential growth phase. The possibilitythat the receptivity of the cells to auxin changed during thegrowth cycle was examined by measuring the activity of a membrane-boundauxin-binding site. In subcultures grown in a medium with anoptimal auxin concentration the maximum auxin-binding activitywas restricted to the end of the exponential growth phase. Inthe cells cultivated in partially or completely auxin deprivedmedia the auxin-binding activity increased to varying extents.These results probably reflect mechanisms controlling both theintracellular content of free auxin and the sensitivity of thecells to exogenous auxin supply (including auxin binding) withrespect to the cell division and/or growth Key words: Nicotiana tabacum L., plant cell culture, IAA, auxin-binding site, cell division     

Biogenesis of auxin in the coleptile of a semi-brachytic barley, uzu

The coleoptile of a semi-brachytic barley, uzu(Hordeum vulgareL. cv. Akashinriki), elongated ca 1/2 as much as the coleoptileof the contrasting normal form which is isogenic excepting theuz gene. This retarded growth of the uzu coleoptile, as comparedto the normal coleoptile, is not due to changes in the rateof basipetal transport of auxin, neither to the destructionof auxin during transport, nor to sensitivity to auxin. Furthermore,less of the extractable and bound auxins were found in the uzu(uzuz)coleoptile than in the normal(UZUZ)coleoptile, suggesting thatthe retarded growth of uzu coleoptile may be due to less auxinproduction. Apical tips of the normal coleoptile grown under sterile conditionsresponded to both tryptophan and tryptamine, but uzu coleoptiletips responded only to tryptamine. Thus, growth retardationof the uzu coleoptile may be due to lower activity of the enzymewhich converts tryptophan to tryptamine in the uzu coleoptile. (Received August 20, 1973; )     

Transport and Accumulation of IAA-14C in Tumour-forming Nicotiana Hybrids

The polar transport of indol-3yl-acetic acid (IAA-2-¹⁴C) instem explants and decapitated shoots of tumour-prone Nicotianahybrids (2n, 3n, and 4n) was compared with that in the normal,non-tumorous parent species N. glauca and N. langsdorffii. Thetotal uptake of the auxin from donor blocks was greatest inthe hybrids and N. glauca. The velocity of the basipetal movementof IAA-¹⁴C was the same in all species tested, i.e. 8 mm/h.The transport capacity for the hormone, however, was decreasedin the three tumour-prone hybrids. Gas chromatography showedthat between 70 and 90 per cent of the transported auxin waspresent in the form of IAA, between 10 and 30 per cent in theform of indol-3yl-aldehyde (IAld). The basipetal transport exceeded the acropetal transport inyoung (third) intemodes of all plants studied, whereas in olderstem segments (tenth intenodes) the reverse was found. The polarity of auxin transport was less well expressed in thetumorous hybrids. Blocking the active transport by pre-treatment of stem cuttingswith 2,4-dinitrophenol (2,4-DNP) caused a drastic reductionin the polar IAA-¹⁴C movement; in all plants tested the auxintransport was reduced to the same low level. The accumulation of auxin at the base of cuttings was higherin N. glauca and the 2n hybrid than in N. langsdorffii, i.e.about seven times higher after 1-h and three times higher after12-h transport experiments. The release of ¹⁴C from the cuttinginto an agar receiver block, however, was markedly reduced inthe 2n hybrid, whereas in N. glauca the labelled substancesmoved more freely into the receiver blocks. Differences in the capacity for the accumulation and the releaseof IAA-¹⁴C in hybrid and N. glauca stem tissues were studiedusing decapitated greenhouse plants wounded by incision abovethe fourth internode. Accumulation of the auxin occurred onlyabove the wound-cut in hybrid plants. This observation is consistentwith the view that tumour formation on hybrid stems occurs atsites of wounding. Our data suggest an elevated auxin levelto be present during tumour initiation at these sites. These results on polar transport and accumulation of IAA-¹⁴Cin tumorous Nicotiana plants together with our previous dataon various endogenous auxins suggest that the induction of neoplasticgrowth in tobacco plants is correlated with increased auxinlevels and an accumulation of the hormone at sites of wounding.     

Further characterization of the spontaneous growth response in Zea coleoptile segments

Excised segments of corn (Zea mays L., Bear Hybrid WF 9?38)coleoptiles show a strong "spontaneous" increase in growth rateabout 4.0 hr after excision. The response can be delayed about2 hr using a brief (10 min) exposure to IAA during the latentperiod. An established spontaneous growth response can be suppressedby a 30 to 60 min exposure to auxin and does not reappear untilabout 2.5 hr after withdrawal of the hormone. During the 3 hrperiod following withdrawal of exogenous auxin there is a two-foldincrease in magnitude and a three-fold decrease in latent periodof a growth response to a sub-optimal level of auxin. The dataare consistent with the hypothesis that the spontaneous growthresponse is caused by a time-dependent change in sensitivityof isolated tissue to auxin and/or a change in the endogenouslevel of auxin. Apical sections of Zea coleoptiles with the tip intact do notgrow at the rapid rate one might expect of tissue with an endogenousauxin supply. Instead they grow very poorly and exhibit botha weak spontaneous growth response and a poor response to exogenouslysupplied auxin. Indirect evidence suggests that this is dueto the production of a growth inhibitor by the tip. (Received August 3, 1976; )     

Endorsperm Development of Maize Defective Kernel (dek) Mutants. Auxin and Cytokinin Levels

To provide insight into the regulatory roles of auxin and cytokininin endosperm development, defective kernel (dek) mutants ofmaize (Zea mays L.) were examined. The mutants dek6, dek18 anddek 26 had substantially lower indole-3-acetic acid (IAA) levelsthan wild-type counterparts while dek8, dek12 dek24 had higherIAA levels than wild-type counterparts. The mutant dek6 hada somewhat lower zeatin level than its wild-type counterpart.The mutation-induced changes in IAA levels and effects of exogenously-appliedauxin were not consistently related to changes in numbers ofnuclei per endorsperm; however, there was a correspondence betweenauxin level in endosperm and nuclear diameter, which is proportionalto the extent of post-mitotic DNA synthesis (endoreduplication).We conclude that auxin may be involved in stimulating endoreduplication.Copyright1993, 1999 Academic Press Zea mays L., maize, defective kernel mutant, dek, auxin, cytokinin, hormone, ELISA, cell number, cell division, endopolyploidy, endoreduplication, DNA amplification     

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; )     

Latitudinal Differences in Coconut Foliar Spiral Direction: A Re-evaluation and Hypothesis

Davis and Davis (Mathematical Modelling8: 730–733, 1987)have previously concluded, based on data collected from 42 locationsaround the world, that the ratio of right-handed to left-handedpalm trees (Cocos nuciferaL.) exhibits hemispheric differences:left-handed palms are in the majority in the Northern Hemisphere,and right-handed palms are in the majority in the Southern Hemisphere.Weighted and unweighted linear regression analyses of theirdata reveal that the skewing quotient [(left-handed—right-handed)/total]is better correlated with magnetic (dip) latitude than withgeographic or geomagnetic (centred dipole) latitude. The hypothesisis advanced that latitude-dependent biases in foliar spiraldirection may be associated with the temporally varying componentof the earth's magnetic field. It is known that changes in thecurrent strength of the ionospheric dynamo induce clockwiseearth currents in the Northern Hemisphere and counterclockwiseearth currents in the Southern Hemisphere. The hypothesis isadvanced that these earth currents, which are measurable intrees, bias the diffusion of auxin (or auxin transport proteins)in young embryos such that left-handed trees are produced preferentiallyin the Northern Hemisphere and right-handed in the SouthernHemisphere. Some tests of the Induced Current Hypothesis areproposed.Copyright 1998 Annals of Botany Company Phyllotaxis, magnetic latitude,Cocos nucifera,coconut palm, foliar spiral, auxin.     

The role of auxin in cell separation in the dehiscence zone of oilseed rape pods

Concentrations of both free and conjugated indole-3-acetic acid(IAA) were studied during development of pod wall, dehiscencezone and seeds of Brassica napus pods. A decrease in auxin contentprior to moisture loss in the pods was observed specificallyin the dehiscence zone, which was correlated with a tissue specificincrease in ß-1,4-glucanase activity. Furthermore,treatment of the pods with the auxin mimic 2-methyl-4-chlorophenoxyaceticacid resulted in a delay of 10 d of ß-1,4-glucanaseactivity and con-comitant cell separation in the dehiscencezone. This indicates that the activity of hydrolytic enzymesinvolved in cell separation in the dehiscence zone is regulatedby auxin activity. Comparison of parthenocarpic pods with seeded pods pointed tothe seeds as the source of IAA. Levels in the dehiscence zoneof these pods were low over the entire sampling period, whilecell separation in the dehiscence zone was delayed by about4 d. These results indicate that a low level of auxins in thedehiscence zone is necessary for dehiscence to take place, butother factors may also be important. Key words: Brassica napus, pod dehiscence, auxin, cellulase     

Relation of special RNA to sensitivity of yeast strains to auxin

RNA functional in auxin action was studied in the followingfour strains of yeasts differing in their sensitivity to auxin:KV2, diploid strain of Saccharomyces ellipsoideus irresponsiveto auxin but made responsive by gibberellic acid treatment;N55, auxinresponsive mutant derived from KV2; A2-0, diploidstrain of 5. cerevisiae irresponsive even when gibberellic acidis given; and A2–N102, responsive mutant derived fromA2–0. The RNA fraction extracted from N55 and partitionedin a phenol layer sensitized KV2 to auxin. This kind of RNAwas not detected in KV2 cells. The functional RNA from N55 wasof low molecular weight, as was functional RNA isolated fromgibberellic acid-treated Jerusalem artichoke tuber. Gibberellicacid-treated KV2 cells have been known to contain RNA whichfunctions similarly to the RNA mentioned above. Both A2–0and A2–N102 contained RNA which made KV2 cells responsiveto auxin. Some factors other than functional RNA may determinethe difference in the sensitivity to auxin between A2–0and A2–N102. (Received August 20, 1968; )     

Changes in o-Diphenol Oxidase During Fibre Development in Cotton

Quantitative and electrophoretic changes in o-diphenol oxidase(o-diphenol: O₂ oxidoreductase, E.C. 1 10,3.1) were studiedduring the entire period of cotton (Gossypium arboreum L. cv.Sanjay) fibre development. A significant increase in o-diphenoloxidase activity was recorded during the fibre initiation phaseand it is suggested that a shift in redox balance towards oxidationmay play an important role in fibre initiation. Low o-diphenoloxidase activity during elongation and its high activity duringthe phase of secondary thickening, together with isoenzyme patterns,suggest an important role of this enzyme in cotton fibre development.The role of o-diphenol oxidase in relation to auxin turnoverand redox balance is discussed. Gossypium arboreum, cotton, fibre development, o-diphenol oxidase, redox balance, auxin turnover     

Histological responses of isolated leaves to hormones applied across a transverse gradient

The responses of petiolar tissue of isolated leaves of Argyreianervosa to hormones were investigated by maintaining a transversegradient of auxin and auxin antagonist or auxin and gibberellin.The auxin used was ß-indolylbutyric acid (IBA) andthe auxin antagonist applied was maleic hydrazide. Gibberellicacid was also applied simultaneously with IBA. The experimentwas designed in such a way that while the solution of one hormonewas applied internally to the petiole with a capillary tube,the external surface of the petiole came in contact with thesolution of the other hormone. Wounding was caused in the pith by inserting a capillary tube.The division of the parenchymatous cells bordering the woundwas greater and cell dimension was less when auxin was appliedinternally but cell division was restricted and cell dimensionincreased when the auxin antagonist was applied at high concentrations.Root primordia and vascular tissues were formed in the pithwhen the concentration of auxin in the vicinity was greater.But these processes were blocked when the concentration of theauxin antagonist was greater in the neighborhood. The effectof auxin and gibberellin was synergistic in inducing these processes. (Received May 23, 1980; )     

Histological responses of isolated leaves to hormones applied across a transverse gradient

The responses of petiolar tissue of isolated leaves of Argyreianervosa to hormones were investigated by maintaining a transversegradient of auxin and auxin antagonist or auxin and gibberellin.The auxin used was ß-indolylbutyric acid (IBA) andthe auxin antagonist applied was maleic hydrazide. Gibberellicacid was also applied simultaneously with IBA. The experimentwas designed in such a way that while the solution of one hormonewas applied internally to the petiole with a capillary tube,the external surface of the petiole came in contact with thesolution of the other hormone. Wounding was caused in the pith by inserting a capillary tube.The division of the parenchymatous cells bordering the woundwas greater and cell dimension was less when auxin was appliedinternally but cell division was restricted and cell dimensionincreased when the auxin antagonist was applied at high concentrations.Root primordia and vascular tissues were formed in the pithwhen the concentration of auxin in the vicinity was greater.But these processes were blocked when the concentration of theauxin antagonist was greater in the neighborhood. The effectof auxin and gibberellin was synergistic in inducing these processes. (Received May 23, 1980; )