Gravitropism and Phototropism of Maize Coleoptiles: Evaluation of the Cholodny-Went Theory Through Effects of Auxin Application and Decapitation

It was investigated whether or not gravitropism and phototropismof maize (Zea mays L.) coleoptiles behave as predicted by theCholodny-Went theory in response to auxin application, decapitationand combinations of these treatments. Gravitropism was inducedat an angle of 30° from the vertical, and phototropism,by a pulse of unilateral blue light. Either tropism of the coleoptilewas inhibited by IAA, applied as a ring of IAA-lanolin pasteto its sub-apical part, and by decapitation. The dose-responsecurves for the effects of applied IAA on tropisms and growthof intact coleoptiles as well as the time courses of tropismsinduced in decapitated coleoptiles could be explained by thethree conclusions in the literature: (1) the tip of the coleoptileis the site of auxin production, (2) lateral translocation ofauxin in gravitropism occurs along the length of the coleoptile,and (3) lateral translocation of auxin in phototropism occursin the coleoptile tip. By examining the effects of decapitationmade at different distances from the top and of IAA appliedto the cut surface of decapitated coleoptiles, it was indicatedthat auxin is produced in the apical 1 mm zone of an intactcoleoptile and that lateral auxin translocation for phototropismtakes place in an apical part that somewhat exceeds the zoneof auxin production. (Received October 14, 1994; Accepted December 26, 1994)     

High-resolution measurement of growth during first positive phototropism in maize†

Abstract Growth redistribution which occurs as a result of phototropic stimulation was studied in red light-grown, maize (Zea mays L.) seedlings. The pattern of elongation of small areas (0.1mm²) of coleoptile epidermis on intact plants was analysed from time-lapse, photomicrographic records. Growth following unilateral, pulse irradiation with blue light was depressed on the illuminated side and was stimulated on the shaded side. The time at which the change in growth rate occurred, on both illuminated and shaded sides, was significantly earlier in apical patches than it was in basal patches. Both kinds of change in the growth rate (stimulation and depression) occurred rapidly such that a new, constant growth rate was often established within five minutes. Micrographic, time-lapse records were also obtained of growth changes induced by sub-apical, unilateral application of a spot of an indole-3-acetic acid (IAA) and lanolin mixture. Growth on the side of the coleoptile to which IAA had been applied was similar to the growth on shaded sides of phototropically stimulated plants. The distance between apical and basal patches and the elapsed time between their changes in growth rate gave a velocity at which the growth response moved basipetally. Calculation of this velocity for blue light and auxin treatment gave values that were not significantly different. Thus, basipetal movement of a transverse auxin gradient could mediate growth changes that cause curvature of the coleoptile towards first positive fluences of blue light.     

The Geoelectric Effect in Plant Shoots: III. DEPENDENCE UPON AUXIN CONCENTRATION GRADIENTS AND AEROBIC METABOLISM

The development of the geoelectric effect has been followedin Zea coleoptiles with a flowing-solution electrode system,and its dependence upon auxin concentration gradients and aerobicmetabolism assessed. A symmetrical source of IAA can effectively replace the coleoptiletip in allowing the geo-electric potential to occur. The diffusatefrom coleoptile tips, when applied asymmetrically to the apexof a vertical decapitated coleoptile, generates a potentialdifference across the coleoptile indistinguishable from thatinduced by the asymmetrical application of IAA. Asymmetricalapplication of IAA to vertical Avena and Zea coleoptiles andHelianthus hypocotyls induces closely similar responses. Neither the geoelectric effect nor a geotropic response developswhen intact Zea coleoptiles are placed horizontally after beingdeprived of oxygen, but they both occur when an aerobic atmosphereis restored. The lateral potential difference induced by theasymmetrical application of IAA to the apex of a vertical coleoptiledoes not occur under anoxic conditions. With a static-drop electrode system and a decapitated Zea coleoptile,a potential difference develops immediately after reorientationof the coleoptile into the horizontal position, and attainsa maximum value after about 10 min. This potential differencecan be further increased by the asymmetrical application ofIAA to the lower half of the apical cut surface of the coleoptile. Our data support the view that both the geoelectric potentialand the geotropic response are due to the IAA concentrationgradient which arises from the lateral transport of this substancefrom the upper to the lower half of the horizontal shoot. Theyalso bear out our previous conclusions that the ‘geoelectricpotential’ observed with static-drop electrodes and anintact shoot, is the resultant of two processes. The first isa physical phenomenon arising in the electrodes, or betweenthe electrodes and the plant tissue, and the second arises inthe living tissues of the shoot as the result of gravity-inducedchanges in auxin distribution.     

Auxin-Growth Relationships in Maize Coleoptiles and Pea Internodes and Control by Auxin of the Tissue Sensitivity to Auxin

Growth of a zone of maize (Zea mays L.) coleoptiles and pea (Pisum sativum L.) internodes was greatly suppressed when the organ was decapitated or ringed at an upper position with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) mixed with lanolin. The transport of apically applied ³H-labeled indole-3-acetic acid (IAA) was similarly inhibited by NPA. The growth suppressed by NPA or decapitation was restored by the IAA mixed with lanolin and applied directly to the zone, and the maximal capacity to respond to IAA did not change after NPA treatment, although it declined slightly after decapitation. The growth rate at IAA saturation was greater than the rate in intact, nontreated plants. It was concluded that growth is limited and controlled by auxin supplied from the apical region. In maize coleoptiles the sensitivity to IAA increased more than 3 times when the auxin level was reduced over a few hours with NPA treatment. This result, together with our previous result that the maximal capacity to respond to IAA declines in pea internodes when the IAA level is enhanced for a few hours, indicates that the IAA concentration-response relationship is subject to relatively slow adaptive regulation by IAA itself. The spontaneous growth recovery observed in decapitated maize coleoptiles was prevented by an NPA ring placed at an upper position of the stump, supporting the view that recovery is due to regenerated auxin-producing activity. The sensitivity increase also appeared to participate in an early recovery phase, causing a growth rate greater than in intact plants.     

Physiological evidence that the primary site of auxin action in maize coleoptiles is an intracellular site

To locate functionally the primary site of auxin action in growing cells, the pool of auxin relevant to induction of growth in maize (Zea mays L.) coleoptile sections was determined. A positive correlation was consistently noted between growth and intracellular levels of indole-3-acetic acid (IAA), i.e. growth appears to be relatively independent of the external level of IAA. N-1-Naphthylphthalamic acid (NPA), a potent inhibitor of auxin transport, was used to enhance accumulation of IAA in coleoptile cells. From the use of NPA, it is shown that: 1) increasing the accumulation of IAA in cells, while the external concentration is held constant, resulted in a concomitant increase in growth, and 2) blocking the exit of IAA from cells with NPA sustained an IAA-induced growth response in the absence of externally applied IAA. Furthermore, the absence of any alterations in auxin binding to microsomal fractions by NPA indicates that the action of NPA in causing enhancement of auxin-induced growth is based upon its inhibition of efflux of IAA from the cells. This research was supported by National Science Foundation grant No. DMB 8515925. The careful assistance of Laurie Brulport is gratefully acknowledged.     

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.     

Correlative Inhibition of Lateral Bud Growth in Phaseolus vulgaris L. Effect of Application of Indol-3yl-Acetic Acid to Decapitated Plants

In view of the variation in the ability of indol-3yl-aceticacid (IAA) to prevent lateral bud growth on decapitated plantsvarious factors which might influence the response have beeninvestigated in Phaseolus vulgaris L. The effectiveness of IAAapplied in lanolin varied from experiment to experiment. Factorsthat altered the response included the time of year, the concentrationand quantity of IAA, the age of the plant, the type of lanolinand the region of application. In many instances IAA, at a concentrationbelieved to mimic the auxin relations of the intact plant, cancompletely replace the main shoot with respect to the correlativeinhibition of lateral bud growth. The evidence for the involvementof IAA as the primary determinant in apical dominance in P.vulgaris is summarized.     

Effect of {alpha}-Tomatine on the Response of Wheat Coleoptile Segments to Exogenous Indole-3-acetic Acid

A concentration of 10^–5 M tomatine had no effect on leakagefrom, or elongation of, wheat coleoptile segments, but consistentlyreduced IAA-enhanced extension growth by c. 50 per cent. Therewas no evidence of chemical interaction between the alkaloidand the auxin in solution, and IAA action was not affected bypre-treatment for up to 3 h with 10^–5 M tomatine. Studieswith [2-¹⁴C]IAA revealed that 10^–5 M tomatine did notinhibit uptake of auxin into segments. The effect of pre-treatingsegments for up to 3 h with IAA could be virtually nullifiedby 10^–5 M tomatine, as could also IAA-induced changesin properties of coleoptile cell walls. Results are discussedin relation to the ability of tomatine to disrupt membrane functionand to current hypotheses implicating membranes in the primaryaction of auxin.     

Methyl jasmonate induces the formation of secondary abscission zone in stem of Bryophyllum calycinum Salisb

Methyl jasmonate (JA-Me) at a concentration of 0.5 % induced the formation of secondary abscission zone and senescence in several types of stem explants (only internode segment, internode segment with nodes and without leaves, internode segment with nodes and debladed petioles) of Bryophyllum calycinum when it was applied in various places of the stem or the debladed petiole as lanolin paste. In the presence of small leaves in stem explants methyl jasmonate also induced the formation of secondary abscission zone and senescence but the presence of larger leaves completely inhibited methyl jasmonate-induced processes. Auxin, (indole-3-acetic acid, IAA), at a concentration of 0.1 % extremely prevented the formation of secondary abscission zones and senescence in the stem tissues induced by methyl jasmonate. Similar relationship between auxin and methyl jasmonate to induce the formation of secondary abscission zone and senescence was found in decapitated shoot of the intact plant. Mechanisms of the formation of secondary abscission zone are also discussed in terms of the interaction of methyl jasmonate with auxin.     

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     

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; )     

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.     

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     

Effect of laterally applied gibberellin A4/7 on cambial growth and the level of indole-3-acetic acid in Pinus sylvestris shoots

Gibberellin A_4/7 (GA_4/7) was applied in lanolin or ethanol around the circumference at the midpoint of the previous-year terminal of dormant Pinus sylvestris seedlings. After cultivating the seedlings under environmental conditions favorable for growth for up to 10 weeks, cambial growth was measured as the radial widths of xylem and phloem, and the level of indole-3-acetic acid (IAA) was determined by combined gas chromatography-mass spectrometry using [¹³₆](IAA) as the internal standard. In intact seedlings, both 1 mg GA_4/7 g^?1 lanolin and 50 mg GA_4/7 I^?1 ethanol increased phloem production and the cambial region IAA level in the current-year terminal, without significantly altering its longitudinal growth. In the previous-year terminal, 1 mg GA_4/7 g^?1 lanolin promoted phloem production at the application point and increased the cambial region IAA level above this point, whereas 50 mg GA_4/7 I^?1 ethanol stimulated the production of both xylem and phloem at the treatment site and elevated the cambial region IAA level beneath it. Laterally applied GA_4/7 at 50 mg I^?1 ethanol stimulated xylem and phloem production in debudded previous-year terminals treated at the apical cut surface with 1 mg IAA g^?1 lanolin, but not in those treated with plain lanolin. However, the promotion of cambial growth in debudded terminals treated apically with 1 mg IAA g^?1 lanolin and laterally with 50 mg GA_4/7 I^?1 ethanol was not associated with an elevated IAA content in the cambial region. The results indicate that exogenous GA_4/7 can promote xylem and phloem production provided an IAA source is present, and that it or a metabolic product acts directly, rather than indirectly by stimulating longitudinal growth and/or raising the cambial region IAA level.     

Sources of Free IAA in the Mesocotyl of Etiolated Maize Seedlings

Sources of free indole-3-acetic acid (IAA) for the mesocotyl of intact etiolized maize ((Zea mays L.) seedlings are evaluated. The coleoptile unit, which includes the primary leaves and the coleoptilar node, is the main source of free IAA for the mesocotyl. The seed and the roots are not immediate sources of IAA supply. Dependence of the apical growing region of the mesocotyl on the coleoptile unit as a source of free IAA is almost total. One-half or more of the supply of IAA comes from the coleoptile tip, the rest mainly from the primary leaves. Removal of the coleoptile tip results in inhibition of mesocotyl elongation. The hypothesis that growth of the mesocotyl is regulated by auxin supplied by the coleoptile is supported. Conjugated forms of IAA appear to play little part in regulating the levels of free IAA in the shoot.     

Plasmodesmata, Tropisms, and Auxin Transport

Attempts were made to disrupt the plasmodesmata between oatcoleoptile cells (Avena saliva L. cv. Victory) by severe plasmolysis.Coleoptiles, allowed to regain turgor after plasmolysis, wereable to execute geotropic and phototropic curvatures and segmentswould grow in response to applied auxin. In coleoptiles similarlytreated, studies with [¹⁴C]IAA have shown that longitudinal,basipetal transport of auxin still takes place and, as in controls,IAA is preferentially redistributed laterally within coleoptilesorientated horizontally. Physical continuity of the symplast of oat coleoptile cellsmay not always be disrupted by severe plasmolysis. Nevertheless,functional continuity appears to be interrupted. Despite this,all the processes involved in the execution of tropistic curvaturesremain intact, including transport of hormones. Plasmodesmatalcontinuity between oat coleoptile cells appears not to be anecessary requirement for auxin transport.     

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.     

Effect of morphactin on stem growth in relation to auxin in precooled rooted tulip bulbs

Effect of morphactin IT 3456, an auxin transport inhibitor, on tulip stem elongation induced by indole-3-acetic acid (IAA) was investigated. Tulip stem growth induced by IAA 0.1 % in lanolin paste applied on the top internode after excision of flower bud and removal of all leaves was greatly inhibited by 0.2 % morphactin IT 3456 applied on the 4th, 3rd, 2nd and 1st internode. The inhibitory effect of the morphactin on tulips stem growth promoted by IAA was restored by additional application of IAA below the morphactin treatment place. Morphactin inhibited also the growth of all internodes induced by flower bud in the absence of leaves. These results suggest a crucial role of auxin in the control growth of all internodes in tulip stem.     

Effects of Morphactin on Indol-3yl-acetic Acid Transport, Growth, and Geotropic Response in Cereal Coleoptiles

The effects of the morphactin 2-ehloro-9-hydroxyfluorene-9-carboxylicacid methyl ester [CFM] on growth, geotropic curvature and transportand metabolism of indol-3yl-acetic acid [IAA-5-³H] in the coleoptilesof Zea mays and A vena saliva have been investigated. A strongcorrelation has been found to exist between the inhibition ofthe geotropic response and the inhibition of auxin transport.CFM supplied at concentrations sufficient to abolish auxin transporthas been shown to promote the elongation of Zea, but not ofAvena, coleoptile segments. CFM does not change the patternof metabolism of IAA in Zea coleoptile segments. In these segmentsIAA is metabolized when its concentration is high, but the radioactivitytransported basipetally, or laterally in geotropically stimulatedcoleoptiles, is virtually confined to the IAA molecule. Radioactivityexported into the basal receiver blocks is wholly confined toIAA. It is concluded that CFM inhibits the geotropic responsein coleoptiles by suppression of the longitudinal and lateralauxin transport mechanisms. The growth-promoting propertiesof this substance cannot be linked with its effects on eitherauxin metabolism or transport.     

Inside or outside? Localization of the receptor relevant to auxin-induced growth

The localization of the auxin receptor relevant to the control of elongation growth is still a matter of controversy. Auxin-induced elongation of maize coleoptile segments was measured by means of a high resolution auxanometer. When indole-3-acetic acid (IAA) was removed from the bathing solution, a rapid cessation of auxin-induced elongation was detected. This decline was delayed when the auxin efflux carrier was blocked by the phytotropins naphthylphthalamic acid (NPA) and pyrenoylbenzoic acid (PBA) or by triiodobenzoic acid (TIBA). The IAA concentration in NPA-pretreated segments was 2–3 times higher than in NPA-free controls 35 min after the removal of IAA in the bathing medium.
A similar rapid drop of growth after removal of auxin was observed for the rapidly-transported synthetic auxin, naphthaleneacetic acid (NAA). When the auxin efflux was blocked, growth induced by NAA was sustained much longer than IAA-stimulated elongation.
In comparison with NAA, the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) is known to be excreted very slowly by the efflux carrier. 2,4-D-induced growth remained at a stimulated level when the auxin was washed off, even in the absence of any auxin efflux inhibitor. We conclude from these results that the presence of intracellular auxin is a necessary and sufficient condition for sustained auxin-induced elongation growth, at least for the phases during the 2 h after its application. Consequently, we postulate the existence of an intracellular auxin receptor relevant to the control of growth.