The Geoelectric Effect in Plant Shoots:: IV. INTER-RELATIONSHIP BETWEEN GROWTH, AUXIN CONCENTRATION AND ELECTRICAL POTENTIALS IN ZEA COLEOPTILES

Incubation of Zea coleoptiles in 0.5 M mannitol totally inhibitsgrowth and geotropic curvature, but does not affect the developmentof the geoelectric effect. This pre-treatment also inhibitsthe curvature induced by the asymmetrical application of IAAto the apical end of decapitated vertical coleoptiles, but itdoes not prevent the IAA from giving rise to an electropotentialdifference between the two sides of the coleoptile. Neitherthe normal geoelectric effect, nor the auxin-induced potentialdifference in vertical coleoptiles, can therefore arise as theresult of the different rates of cell extension in the two halvesof the organ. They must be the result of the change of IAA concentrationaffecting some other aspect of the cell's physiology or metabolism. The abolition of the electrical responses in coleoptiles whichhave been plasmolysed in 1.0 M mannitol strongly suggests thatboth longitudinal and lateral transport of IAA are severelydepressed by this degree of plasmolysis. Asymmetrical application of 10^-5 M mersalyl and several othersubstances to the apical end of a decapitated vertical coleoptilegave rise to a marked electropotential difference between thetwo sides of the coleoptile, the side beneath the donor beingpositively charged with respect to the other side. Mersalyldoes not promote the growth of Zea coleoptiles. These resultsprovide additional evidence that the electropotentials do notarise from differential growth, and suggest that such substances,especially the diuretics used in clinical medicine, may provideuseful tools in the further study of the induction of surfaceelectropotentials in plant tissues at the cellular level.     

The lateral transport of IAA in intact coleoptiles of Avena sativa L. and Zea mays L. during geotropic stimulation

Summary Movement of IAA was studied in excised coleoptile apices and whole seedlings of Zea mays L. and Avena sativa L. during geotropic stimulation. A micropipette technique permitted the application of [5-³H]IAA at predetermined points on the coleoptiles with minimal tissue damage.When [5-³H]IAA was applied to the upper side of a horizontal excised Zea coleoptile, about 60% of the recoverable radioactivity had moved into the lower half after 2 h. In contrast, when application was made to the lower side of a horizontal excised coleoptile, only 4% of the radioactivity migrated to the upper half. There was, thus, a net downward movement of 56%. Similar patterns of distribution were found for radioactivity in both the tissue and the basal receiver blocks. In horizontal shoot tissues of intact Zea seedlings a net downward movement of about 30% of the recoverable radioactivity occurred after 1 h of geotropic stimulation. Comparable experiments with Avena indicated a net downward movement of 6–12% in excised apices of coleoptiles and in the intact shoot. In both Zea and Avena chromatographic analyses of tissue and receiver blocks indicated that the movement of radioactivity reflected that of IAA.In Zea coleoptiles, the lateral migration of radioactivity after 2 h was 3 to 4 times greater in the apical tissues than in the basal tissues. A significant net downward movement of radioactivity was detected after 10 min of geotropic stimulation in the extreme apex of Zea coleoptiles but not in the more basal regions.These experiments show that downward lateral transport of IAA occurs in intact shoots of Zea and Avena seedlings upon geotropic stimulation. Lateral transport of IAA had previously been demonstrated only in sub-apical segments of Zea coleoptiles.     

The Geoelectric Effect in Plant Shoots: I. THE CHARACTERISTICS OF THE EFFECT

A comparative study has been made of the geoelectric effectin Helianthus hypocotyls and Zea coleoptiles using two electrodesystems. With the static-drop electrode system a potential differencedeveloped between the upper and lower surfaces of the shootsimmediately after they were turned into the horizontal position.The lower surface of the shoot became positively charged withrespect to the upper surface. In non-decapitated shoots thispotential difference continued to increase for at least 20 min,whereas in decapitated shoots no further increase occurred afterabout 10 min from the moment of reorientation. In contrast,with a flowing-solution electrode system no potential differencedeveloped in non-decapitated shoots until about 12 min afterthey were placed in the horizontal position. Thereafter thelower surface became increasingly positively charged with respectto the upper surface for at least a further 12 min. In decapitatedshoots there was no tendency whatsoever for the lower surfaceof the horizontal shoot to become positively charged with respectto the upper surface, even after 25 min in the horizontal position.The static-drop electrode system has an inherent sensitivityto reorientation in a gravitational field; a potential differencedevelops immediately after reorientation, and increases to amaximum value within the first 10 min of reorientation, regardlessof whether or not the electrodes are in contact with plant tissue.The continued increase in the potential difference measuredacross the shoots with the static-drop electrode system, andthe entire development of the potential difference measuredwith the flowing-solution electrode system, are both dependentupon the shoot apex being intact. These facts have enabled usto show that the electro-potential difference measured acrosshorizontally placed non-decapitated tissues with the static-dropelectrode system is the resultant of two distinct processes:(a) an immediate, purely physical electrical effect generatedin the electrodes themselves, and (b) a delayed geoelectriceffect which arises solely in the living tissues of the shootand which is dependent upon the apex of the shoot being intact.     

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)     

The Geoelectric Effect in Plant Shoots: II. SENSITIVITY OF CONCENTRATION CHAIN ELECTRODES TO REORIENTATION

A concentration chain, static-drop electrode system has beenused by several investigators to measure the geoelectric effectin plant shoots. This paper describes investigations of theinherent sensitivity of this electrode system to reorientationwith respect to gravity. When the gelatine plug of the electrodeis made up with distilled water, and the contact solution is0.1 mM KCl, a potential difference develops immediately afterelectrodes in direct contact are rotated through 90° intothe vertical plane. A similar response is found when the contactsolution is 5 mM CaCl₂. Increasing the concentration of thecontact solution, or incorporating KCl, K₂SO₄, or ZnSO₄ intothe gelatine plug, drastically reduces the potential differencedeveloped after reorientation. The potential difference acrosselectrodes in direct contact decreases as the electrodes age.The potential difference measured with these electrodes acrossa decapitated, horizontally placed, hypocotyl of Helianthusarmuus also decreases as the electrodes age. The polarity ofthe charge is reversed as compared with that found when theelectrodes are in direct contact. The kinetic characteristicsof the geoelectric potential difference developed across a non-decapitated,horizontal coleoptile of Zea mays change as the electrodes age.With fresh electrodes the potential develops immediately afterreorientation and continues to increase with time. With 4-day-oldelectrodes, however, no potential difference develops until9 min after the moment of reorientation, but then it increaseswith time. The characteristics of the geoelectric potentialdifference developed with the aged concentration-chain, static-dropelectrodes are similar to those found with several other typesof electrodes which do not themselves have an inherent sensitivityto reorientation with respect to gravity. The results supportour earlier suggestion that the potential difference which apparentlydevelops with the static-drop electrode system, immediatelyafter a shoot is turned through 90° in reality developsin the electrode system itself and not in the plant tissue.The geoelectric effect which arises in the living plant shootbegins to develop approximately 10 min after reorientation.     

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.     

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.     

The Effects of Indole-3-Acetic Acid and 2:4-Dichlorophenoxyacetic Acid on the Growth Rate and Endogenous Rhythm of Intact AvenaColeoptiles

The rates of elongation of the coleoptiles of Avena seedlings,subjected to intermittent immersion in solutions of IAA or 2:4-Dfor various total periods, were determined from measurementsof photographs taken every hour by infra-red radiation. Immersion in 17·5 mg./l. IAA for 1–5 hours causeda large increase in the growth rate followed by a depression.When the seedlings were immersed in 8·75 mg./l. IAA forperiods of 12 or 24 hours the depression was partially overcomeso long as the treatment was continued. Absorption of additionalIAA by the coleoptiles reduced their geotropic sensitivity. Penetration of 2:4-D (sodium salt) into the coleoptiles wasslower than that of IAA and the resulting stimulation of thegrowth rate was less, particularly in unbuffered solutions.After the treatment the growth rate declined slowly to aboutthe normal value. Results with coleoptiles were very similar to those previouslyobtained with rhizomes of Aegopodium and suggest that inhibitionof growth following stimulation by IAA may be of general occurrence.Possible causes of the inhibition are discussed and a comparisonis made between the results with intact coleoptiles and observationsmade by others on coleoptile sections. Temporary immersion of the seedlings in auxin solutions depressedthe rate of elongation of the primary leaf while it increasedthat of the coleoptile. It caused little disturbance of theendogenous rhythm induced by change from light to darkness.The suggestion that such rhythms can be explained in terms ofvariation in concentration of IAA-oxidase is not supported.     

Geotropic curvature of decapitated Avena coleoptiles after application of tips of maize roots,tips of Avena coleoptiles,indoleacetic acid,or abscisic acid

Isolated Avena coleoptiles were decapitated at different distances from the tip and then placed horizontally, after which the geotropic curvature was measured. No geotropic curvature could be detected during the first 3 h. Later, upward curvature occurred which was found to depend inversely on the length of the decapitated tips. When the tips of maize roots or Avena coleoptiles were placed on the cut surface of decapitated Avena coleoptiles, the coleoptiles showed a significantly stronger upward curvature as compared to controls which had been provided with agar blocks on the cut surface. The same upward curvature was found with decapitated coleoptiles provided with agar blocks containing 10^-6 or 10^-7 M indoleacetic acid (IAA). After application of abscisic acid (ABA) at concentrations of 10^-6 and 10^-8 M to the decapitated coleoptiles, the curvature observed was not different from that of the controls; at higher concentrations of ABA the curvature was found to be lower than that of the controls. It is concluded that root tips secrete a substance which may replace the effect of IAA in coleoptiles. The results are discussed in view of the validity of the Cholodny-Went hypothesis for the geotropic reaction of roots.Abbreviations ABA abscisic acid - IAA 3-indoleacetic acid     

A Comparison between Geotropism and Geoelectric Effect in Pisum sativum and Its Mutant ageotropum

The auxin concentration in roots of Pisum sativum ageotropum was examined by three indirect methods:

• 1) Supply of auxin before geotropic stimulation;
• 2) Lateral placing of the root tip;
• 3) Inhibiting the auxin transport in half of the root.

All the results indicated supraoptimal auxin concentration. When decapitated ageotropum roots were supplied with 1.5 mm long tips from normal Pisum roots their geotropic reactivity was almost restored. The geoelectric potential of stems of Pisum sativum and its mutant ageotropum was measured. In ageotropum stems the geoelectric potential was less and the geotropic reaction appeared later than in the normal stems.     

The Role of the Coleoptile Apex in Controlling Organ Elongation: I. THE EFFECTS OF DECAPITATION AND APICAL INCISIONS

It has been widely accepted for over 50 years that the elongationrate of a coleoptile is dependent on the supply of auxin fromthe apex. The original coleoptile decapitation experiments whichprovided support for this view have been repeated but the measurementsof coleoptile elongation were made with greater temporal andspatial precision. The experiments confirm that Avena and Zeacoleoptile elongation is retarded by decapitation but the locationand timing of the growth rate changes are not consistent withthe hypothesis that decapitation reduces growth rate solelyby removing the major supply of auxin. Evidence is presentedthat wounding is the prime cause of the effects of decapitation.Data are also presented showing that the recovery of growthrate of coleoptiles after decapitation or wounding is not dearlyassociated with any events near the cut surface and hence thetraditional explanation of this phenomenon (‘regenerationof the physiological tip’) is misleading. Key words: Coleoptile, decapitation, apex     

IAA transport during the phototropic responses of intact Zea and Avena coleoptiles

Summary Transport of indolyl-3-acetic acid (IAA) was studied during the phototropic responses of intact shoots and detached coleoptiles of Zea mays L. and Avena sativa L. The use of a high specific activity [5-³H]IAA and glass micropipettes enabled asymmetric application of the IAA to be made to individual coleoptiles with minimal tissue damage.A unilateral stimulus of 2.59×10^-11 einstein cm^-2 of blue light, probably in the dose range of the first positive phototropic response, caused significant net lateral movement of radioactivity from [5-³H]IAA away from the illuminated side of intact shoots and detached coleoptile apices of both Avena and Zea. The magnitude of the net lateral movement was 15.3% in Zea seedlings and 12.3% in Avena seedlings. Chromatographic analyses indicated that the movement of radioactivity reflected that of IAA. A phototropic stimulus of 1.24×10^-7 einstein cm^-2, which was probably in the second positive dose range, caused significant lateral movement of radioactivity in intact shoots and detached coleoptiles of Zea but not of Avena.In intact Zea seedlings, neither phototropic dosage affected the longitudinal transport of IAA. In intact Avena seedlings, first positive stimulation inhibited longitudinal transport only when the IAA was applied to the illuminated side of the coleoptile, but second positive stimulation inhibited basipetal movement of IAA regardless of the side of application.Exposing the intact seedlings to red light before phototropic stimulation abolished lateral transport after a first positive stimulus in Zea and in Avena.Phototropic stimulation can thus induce a lateral transport of IAA towards the shaded side of the coleoptiles of both Zea and Avena seedlings, and can affect longitudinal movement of IAA in the coleoptile of Avena. However, since phototropic curvature was observed under certain conditions in the absence of either of these effects, the extent to which they are involved in the induction of asymmetric growth in a stimulated coleoptile has yet to be resolved.     

Auxin Transport in Zea mays Coleoptiles II. Influence of Light on the Transport of Indoleacetic Acid-2-C

The effect of bilateral irradiation with white light (1000 Meter Candle Sec) on the basipetal transport of auxin has been investigated. Illumination of either the intact shoot or the excised coleoptile tip of the Zea seedling, decreased the amount of diffusible auxin obtained from the tip, and decreased Avena curvature response to unilaterally applied indoleacetic acid. Irradiation of the intact Zea seedling did not affect the absorption of ¹⁴C-labeled indoleacetic acid from an agar block subsequently placed on the decapitated coleoptile. However, light caused a significant decrease in the amount of labeled auxin basipetally transported, without affecting materially the velocity of that transport. These and other observations are interpreted as support for the hypothesis that the primary hormonal phenomenon in first-positive phototropism is a light-induced impairment in the basipetal transport of auxin.     

Lateral electrical potential following asymmetric auxin application to maize coleoptiles

Following asymmetric application of indoleacetic acid to maize (Zea mays L.) coleoptiles the early time course of changes in lateral electrical potential was externally monitored with static-drop electrodes. First, an early negative potential change of ca.-1 mV was measured at the surface on the side of a strong auxin application. This negative auxin effect ended after ca. 15 min and was followed by a strong and lasting auxin stimulation of a positive lateral potential up to +12 mV at the auxin-treated side. The initial auxin effect appeared to depend on the size of the step-up in auxin concentration.     

The Effects of Externally Applied 3-indolylacetic Acid on Phototropic Induction and Response in the Coleoptile of Avena

The effects of intermittent immersion of Avena seedlings insolutions of IAA on the response of the coleoptiles to unilateralillumination in the region of that producing the second positivecurvature were studied by means of automatic time-lapse photographywhich enabled the growth-rate and curvature to be recorded simultaneously. Phototropic induction occurred even after the coleoptiles hadabsorbed sufficient IAA from a 10^-4 M. solution to raise theirrate of elongation to about twice the normal value. Phototropiccurvature, which had been temporarily inhibited by a curvaturein the opposite direction induced by the IAA, became evidentas soon as this curvature had ceased to operate. In coleoptiles, supplied with IAA after the commencement ofa phototropic curvature, the response was temporarily suppressed.It was resumed as soon as the effects of the exogenous IAA haddisappeared. The ability of the coleoptiles to produce a slight phototropicresponse persisted even when their growth-rate had been greatlyreduced by previous removal of the endosperm. Increasing thegrowth-rate by supplying the starved seedlings with IAA or sucrose,separately or together, failed to increase the response. Decapitation did not prevent phototropic induction, but delayedthe onset of the response. Application of IAA by intermittentimmersion in a 0.1 mg./l. solution, after the decapitated coleoptileshad been exposed to unilateral illumination, increased the rateof growth but reduced the response. The results suggest that in these experiments phototropic inductionwas not mediated by any direct action of light on the displacement,inactivation, or rate of synthesis of an endogenous auxin. Theyare in agreement with the hypothesis that the stimulus causedan asymmetrical distribution of a co-factor of auxin.     

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 Peeling on IAA-induced Growth in Avena Coleoptiles

The act of peeling removes the epidermis exclusively from Avenacoleoptiles. Peeling inhibits IAA-induced growth, by inhibitingthe growth of segments incubated in the presence of IAA, andpromoting that of those incubated in water. The magnitude ofthe inhibition of IAA-induced growth is proportional to theamount of epidermis removed. It is shown that neither lateralswelling, wounding, anaerobiosis, nor exposure to supraoptimalconcentrations of IAA cause the inhibition. It is concludedthat in Avena coleoptiles the epidermis regulates the rate ofexpansion of the underlying parenchyma cells and is the principaltarget of IAA-action. Avena sativa L., oat, coleoptile, indol-3-ylacetic acid, auxin, extension growth     

Identification of auxin from Zea coleoptile tips by mass spectrometry

Summary The auxin from Zea coleoptile tips has been identified conclusively as IAA.     

IAA-Induced Growth Responses of Decapitated Corn Seedlings: Indications of Two Apparent Adaptations with a Possible Role in Gravitropism

The vertical growth responses of corn seedlings (Zea mays L. Mo17 × B73) were determined over an 8-hour period. When seedlings were decapitated 3 millimeters from the coleoptile's tip and supplied with indole-3-acetic acid (IAA) in 1.5% agar blocks, the response was dependent both on time and IAA concentration. The dose-response curves changed in shape and magnitude depending on the total time of IAA application. High concentrations (>3.2 × 10⁻⁶ molar) initially produced high relative growth rates that decreased back to the intact rate (0.03 millimeter per hour per millimeter) after 3 hours. Low concentrations (<1.0 × 10⁻⁶ molar), or agar blocks without IAA, resulted in a rapid decrease from the intact rate to a level that stabilized at 0.01 millimeter per hour per millimeter until the growth rate began to recover after 3 to 4 hours. Intermediate concentrations produced responses similar to that of the intact organ, though some features of these responses were unique.

The coleoptile curvature in response to gravity depended upon whether the coleoptiles were intact, decapitated, or decapitated and supplied with IAA. Coleoptiles decapitated and not supplied wth IAA showed little or no curvature for 3 hours after decapitation. By this time an adaptation, evoked by the low IAA level, had developed and the coleoptiles began to curve steadily. When 1.0 or 3.2 × 10⁻⁶ molar IAA was supplied, curvature was initiated within the first 30 minutes and reached a maximum rate before decreasing and stopping after 3 to 4 hours. The sequence of events in response to these concentrations was similar to the intact sequence but the curvature rate was reduced to one-third to one-half. A model for the autotropic response involving an auxin concentration-dependent, growth-modulating mechanism capable of two modes of adaptation is described.

     

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; )