Morphology and Hormone Levels of Tobacco and Carrot Tissues Transformed by Agrobacterium tumefaciens II. IAA Biosynthetic Activity of Cultured Carrot Tissues Transformed with Wild-Type and Mutant Ti Plasmids

IAA biosynthetic activity was examined in cultured carrot tissuestransformed with Agrobacterium tumefaciens harboring wild-type,aux^– or cyt^– Ti plasmids. In vitro IAAM hydrolaseactivities in tissues transformed with wild-type, and cyt^–Ti plasmids were 3.09 and 19.82 nmol/g proteins/30 min, respectively,but not detectable when aux^– Ti plasmids were used. Theactivity of IAA biosynthesis, determined by the incorporationof radioactivity into IAA in tissues fed with [¹⁴C]-tryptophan,was 34.13, 10.92 and 32.47 pmol/g fr wt/30 min in tissues transformedwith wild type, aux^– and cyt^– Ti plasmids, respectively.The incorporation of radioactivity into the IAAM fraction wasdetected only in the tissues transformed with wild type andcyt^– Ti plasmids. These results showed that the T-DNAencoded pathway of IAA biosynthesis was active in tissues transformedwith wild-type and cyt^h Ti plasmids, and that the activity ofIAA biosynthesis in those tissues was higher than that in tissuestransformed with the aux^– Ti plasmid. (Received March 16, 1988; Accepted July 31, 1988)     

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)     

Growth Substance Interactions during Uptake by Mesocotyl Segments of Zea mays L.

The effects of various growth substances on the ‘metabolic’uptake of indol-3yl-acetic acid (IAA) by Zea mesocotyl segmentswas investigated using methods of fluorescence spectroscopyand radioactivity assay. 2, 4-Dichlorophenoxyacetic acid (2,4-D) and -(I-naphthylmethylthio)propionic acid (NMSP) exertedno discernible effects on IAA uptake, whereas N-I-naphthylphthalmicacid (NPA) stimulated uptake to some degree. Low concentrationsof 2,3,5-tri-iodobenzoic acid (TIBA) promoted the uptake oflow IAA concentrations, while higher concentrations were decidedlyinhibitory. 2,4-Dinitrophenol (DNP), ioxynil, and bromoxynilalso induced marked inhibition presumably by preventing oxidativephosphorylation. The uptake interactions between these compoundswere examined in relation to concentration and time. In no casewas there evidence of competitive interaction. The inhibitoryeffects of TIBA on IAA uptake were considerably greater thanthose of DNP. SH-enzyme protectors such as BAL and cysteinedid not relieve these inhibitions. The absorption of TIBA-¹³¹Iwas completely unaffected by any concentration of IAA tested.Chromatographic and radio-autographic analysis revealed no detectableproducts of IAA-I-¹⁴C metabolism or degradation in maize mesocotyltissue during the 6-h experimental period and this was not alteredby TIBA treatment. Respiratory decarboxylation of IAA-I-¹⁴Cwas found to be negligible and unaffected by TIBA.     

Uptake, Translocation, and Metabolism of IAA in the Olive (Olea europea): I. UPTAKE AND TRANSLOCATION OF [1-14C] IAA IN DETACHED 'MANZANILLA' OLIVE LEAVES

Absorption, translocation, and decarboxylation of [1–¹⁴C]IAAby excised mature and young olive leaves were studied. The decarboxylationwas considerably more intense in mature leaves than in youngones, while the opposite was true for absorption. The rate ofdecarboxylation was dependent on the presence of peltate scalesof the leaves. The amount of non-biological decarboxylationand the possible effect of bacterial contamination on the systemwere studied and the rate of their involvement is discussed.     

Morphology and Hormone Levels of Tobacco and Carrot Tissues Transformed by Agrobacterium tumefaciens III. Formation of IAA Conjugates in Cultured Carrot Tissues Transformed with Wild-type and Mutant Ti Plasmids

Inactivation of IAA was examined in cultured carrot tissuestransformed with Agrobacterium tumefaciens that harbored wild-type,aux^– or cyt^– Ti plasmids. IAA oxidase activities were similar among the three types oftransformed tissue. Metabolites of IAA were examined by followingthe fate of 3-indolyl-[l-^l4C]IAA. IAA conjugates were detectedin all transformed tissues as well as in hypocotyl segmentsof non-transformed carrot seedlings. The rate of formation ofIAA conjugates was ten times higher in the tissues transformedwith wild-type or cyt^– Ti plasmids than in the tissuestransformed with aux^– Ti plasmids. When the tissues transformedwith aux^– Ti plasmids were cultured on medium that containedIAA for 6 h, the rate of formation of IAA conjugates in thesetissues became as high as that in tissues transformed with wild-typeor cyt^– Ti plasmids. The tissues transformed with wild-type or cyt^– Ti plasmidsnot only synthesize larger amounts of IAA but also convert alarger amount of free IAA to conjugated IAA than do non-transformedand aux^– transformed tissues. Presumably, in carrot, theformation of IAA conjugates decreases the amount of free IAAin the transformed tissues that synthesize large amounts ofIAA and, consequently, the level of free IAA can be maintainedfairly constant. (Received June 2, 1989; Accepted May 23, 1990)     

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

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

Factors Affecting Calcium Transport and Basipetal IAA Movement in Tomato Fruit in Relation to Blossom-end Rot

Factors affecting the uptake and distribution of calcium (Ca)by detached tomato (Lycopersicon esculentum Mill.) fruit wereinvestigated in seven cultivars with different susceptibilitiesto blossom-end rot (BER), a physiological disorder caused byCa deficiency. Plants were grown with different levels of salinityin the root zone or under shade to induce BER. In addition,fruit grown at different salinities were treated with CME, aninhibitor of auxin transport to alter IAA movement. The basipetalmovement of indole-3-acetic acid (IAA) out of detached fruit(i.e. IAA efflux) was determined concurrently with ⁴⁵Ca uptaketo assess the possible involvement of IAA in Ca import or theincidence of BER. High salinity in the root zone during fruitdevelopment decreased both the uptake and distribution of ⁴³Cato the blossom-end of the detached fruit. Shading and the applicationof CME reduced ⁴⁵Ca uptake to a lesser extent. IAA efflux, however,was not consistently reduced by these treatments. Neither theuptake and transport of ⁴⁵Ca within, nor the efflux of IAA from,detached fruit was related to the cultivar susceptibility toBER. The proposed role of IAA on the uptake and distributionof Ca by tomato fruit is assessed. Key words: Tomato, calcium, IAA, blossom-end rot, salinity     

A Model for the Interaction of Low Temperature, ABA, IAA, and CO2 in the Control of Stomatal Behaviour

In the chilling sensitive (C.S.) species Phaseolus vulgarisit was found that at 22 ?C ABA induced stomatal closure butthis effect was dependent on the presence of CO₂. In the absenceof CO₂ the effect of ABA was completely lost. In contrast toABA, the effect of IAA at 22 ?C was to increase stomatal openingas the IAA concentration increased from 10^–2 to 10 molm^–3, and this effect was dependent upon the presence ofCO₂. However, at 5 ?C the action of ABA was reversed and itwas found to induce stomatal opening when fed via the transpirationstream in excised leaves. Similarly, the CO₂ response characteristicswere reversed at low temperatures as removal of CO₂ from theatmosphere caused stomatal closure. However, the effect of IAAat 5 ?C in the presence of CO₂ and with or without ABA was toincrease stomatal aperture with increasing IAA concentration.Significantly, ABA was found to have no effect upon aperturein the presence of CO₂ when IAA was added. The interactive effectsof ABA, IAA, CO₂ and low temperature are discussed in relationto a model proposed by the authors. Key words: IAA, ABA, CO₂, Stomata     

Auxin Transport in Roots: V. EFFECTS OF TEMPERATURE ON THE MOVEMENT OF IAA IN ZEA ROOTS

The effects of temperature on the polar movement of IAA through6-mm and 12-mm segments of Zea mays roots have been investigatedover the range from 1 to 50°C. At all temperatures an acropetal polar movement of IAA predominated,although at low temperatures and at 50°C the 6-mm segmentsshowed a transient basipetal polarity, before the persistentacropetal polarity developed. At 1°C the differences betweenacropetal and basipetal movement of IAA were less distinct thanat the other temperatures. There is, however, a marked metabolically-dependentacropetal movement of IAA through the tissues at 1°C, becausewhen the segments were deprived of oxygen the acropetal movementwas severely reduced while the basipetal movement was reducedto a smaller extent. At 1°C and at 5°C there was alwaysa persistent basipetal polarity of IAA movement through 6-mmand 12-mm segments under anaerobic conditions. The velocity of acropetal movement (mm h^–1) was the samethrough the 6-mm and the 12-mm segments and was markedly affectedby temperature. It increased from 1°C to a maximum valueof 8 mm h^–1 at 31°C and then decreased again at 40and 50°C. The velocity of basipetal movement could be assessedonly at 1 and 5°C at which temperatures it was greater thanthe velocity of acropetal movement, and virtually independentof segment length. The acropetal flux of IAA (cpm h^–1) was much less through12-mm segments than through 6-mm segments. For both lengthsof segment, however, the flux showed a complex relationshipwith ambient temperature, increasing from 1°C to a maximumat 10–15°C, declining to a minimum value at 31°Cand then rising again at 40 and 50°C. The basipetal fluxof IAA could be astimated only at 1 and 5°C at which itwas very much smaller than the acropetal flux. The amount of IAA in the receiver blocks increased linearlywith time at the lower temperatures. At temperatures withinthe range 15°C to about 31°C, however, the amount ofIAA in the receiver blocks began to decline if the transportperiods exceeded a certain length. The time at which this declinein the IAA in the receiver block began was related to the ambienttemperature. Chromatographic analysis indicated one radioactive substancein receiver blocks at the apical end of segments supplied withIAA-1-¹⁴C at the basal end after transport periods of 6 h at25°C, and 72 h at 5°C. The Rf of this substance wasclosely similar to that of the radioactive IAA supplied in thedonor blocks.     

The Metabolism of 3-Indolylacetic Acid in Plant Tissues3

The nature of metabolic products of 3–indolylacetic acid(IAA) extracted from potato tuber disks treated with aeratedIAA solution has been investigated. Two major products, knownat first as ‘V’ and ‘P’ in these studieshave been isolated and ‘V’ has been identified as3-indolylacetylaspartic acid (IacAsp). The rapid uptake of IAA is inhibitited by metabolic poisonssuch as 10^–3 M. cyanide. The maximum mean internal concentrationexceeds the external concentration well–aerated cultures.The mean internal concentration, however only remains for aperiod and then falls off rapidly as a result of extrusion ofabsorbed IAA into the external solution. This extrusion is notinhibited by 10^-3 cyanide; when the mean internal IAA concentrationis 150 µ mol/ml. and the localized IAA concentration musttherefore exceed this value. We conclude therefore that theIAA concentration in the sites where it has accumulated exceedsthe concentration of IAA outside. Uptake of IAA and also its further conversion are inhibitedby indolylacetonitrile and promoted by aspartate, but this promotionis not associated with any gain in amount of indolylacetylaspartate(IacAsp). The data suggest that IacAsp may be formed in tissue from ‘boundIAA’ rather then free IAA. The ‘accelerator ’ found in potato and beans whichhas similar R_F to IAcAsp has been shown definity to be someother substance or substances and not IAcAsp as was at firstthought possible.     

Regulation of Sulphate Transport in a Tropical Legume, Macroptilium atropurpureum, cv. Siratro

When young plants of Macroptilium atropurpureum, cv. Siratrowere deprived of external sulphate (-S plants) growth of shootsand roots continued at rates comparable to those in plants wellsupplied with sulphate (control) for 3 d and 5 d respectively.Dilution of internal sulphur therefore took place and redistributionof sulphur occurred between inorganic and organic forms andbetween roots and younger leaves. Even when S-deficiency limitedgrowth, plants contained 16% of their total sulphur as sulphate,but most of this was retained in old leaves and redistributedslowly to growing zones. The capacity for sulphate uptake increased in roots of –Splants very soon after they were deprived of external sulphate;within 24 h the absorption from 0.25 mol m^–3 SO₄^2–was more than five times that of control roots. Maximum increasedcapacity was reached after 2–3 d stress when the V_maxof system 1 was 1948 nmol h^–1g^–1root fr. wt. in–S plants and 337 nmol h^–1g^–1root fr. wt.in controls. The K^mfor system 1 did not change significantlywith S-stress being between 5–8 µM in both setsof plants. Absorption of L-cysteine was not stimulated by S-stress. There was a close, positive relationship between plant growthrate and the rate at which sulphate uptake capacity was enhancedby withholding sulphate from culture solutions. When –S plants were replaced in sulphate-containing solutiontheir capacity for SO₄^2– declined to the control levelwithin 24 h. Very marked repression of capacity was also foundwhen –S plants were treated with L-cysteine, but therewas no immediate effect with methionine. Roots of this species appear to have a very active system fordegrading L-cysteine to sulphate, 30% of the label in ³⁵S-cysteineabsorbed by roots was recovered in ³⁵SO₄^2– after 20 minor 2 h incubation. By contrast, roots had a very weak abilityto reduce sulphate. When part of the root system was in solution lacking sulphatethere was enhanced uptake of sulphate by other parts which themselveswere amply supplied with sulphate. This is seen as an exampleof compensatory absorption. The response to S-stress is specific and there were no positiveinteractions between S-stress and the absorption of phosphate,or P-stress and the uptake of sulphate. The results are discussed in relation to the close control ofsulphate uptake by internal sulphate concentration, redistributionof forms of sulphur during stress and mobility of sulphate inthe phloem. Key words: Kinetics, Amino-S, Sulpholipid, Repression;, Deficiency     

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 Polarity of Movement of Endogenously Produced IAA in Relation to a Gravity Perception Mechanism

The polarity of radioactive IAA transport in segments of theleaf sheath base of Avena fatua L. is reversed upon their inversionthrough 180° transport towards the apex being greater thantowards the base. Changes in rates of transport leading to thisreversal can be detected within 10–20 min, correlatingwith the timing of statolith movements from the base to theapex of statocyte cells and conforming to a proposed model forthe gravity control of auxin transport. Transport of H³-trytophan-derived H³-IAA was found to undergosimilar changes in polarity upon re-orientation as did exogenouslyapplied H³ or C¹⁴ IAA. It is concluded that the proposed modelalso relates to the movement of endogenously produced IAA.     

The influence of IAA on the uptake of potassium,calcium, magnesium,water absorption and growth in young maize seedlings

The influence of IAA in two concentrations (10⁻⁸M and 10⁻⁵M) on relations between growth, water absorption and cation uptake and accumulation was tested.IAA in a higher concentration retarded growth remarkably. First of all, potassium uptake and water absorption were significantly decreased while the uptake of divalent cations was affected later and less remarkably. 10⁻⁸ M IAA accelerated the growth rate slightly together with acceleration of water absorption and cation uptake. Presented at the International Symposium “Plant Growth Regulators” held on June 18 – 22, 1984 at Liblice, Czechoslovakia.     

The Effects of Mechanical Impedance to Growth on the Levels of ABA and IAA in Root Tips of Zea mays L.

Extraction and analytical methods have been refined and newones devised to allow precise determinations by GC-EC of thelevels of abscisic acid (ABA) and indol-3ylacetic acid (IAA)in samples of maize root tips as small as 1.0 g fr. wt. Seminalroots of 5-d-old maize seedlings grown in normal (bulk density1200 kg m^–3) and compacted (bulk density 1600 kg m^–3)sand/garden loam mixtures have been examined. Seminal rootsfrom compacted soil had an average length of about 40% of thatof control roots and were much thicker. The ABA levels in 10mm tips of impeded roots (c. 25–35 ng g^–1 fr.wt.)did not differ significantly from those of normal root tipson both a fresh and dry weight basis. The levels in 0–1mm tips were approximately double those in the remaining 1–10mm zones. IAA levels were increased by about 3 times in impededroots (176.3 as compared with 52.4 ng g^–1 fr.wt) and itis concluded that this response is likely to be the main causeof the morphological and growth changes brought about by soilcompaction.     

The Uptake of Growth Substances: XIII. DIFFERENTIAL UPTAKE OF INDOL-3YL-ACETIC ACID THROUGH THE EPIDERMAL AND CUT SURFACES OF ETIOLATED STEM SEGMENTS

The patterns of uptake of indol-3yl-acetic acid (IAA-2-¹⁴C)by etiolated stem segments of varying lengths have been examined,employing tissues excised from (a) the first and third internodesof Pisum sativum, (b) the top and base of the hypocotyl of Gossypiumhirsutum, and (c) the mesocotyl of Avena sativa. For all species,concentrations (10^–5–10^–3 M) and times upto 24 h, there is a steady accumulation of radioactivity inthe segments. For equal volumes of tissue uptake is inverselycorrelated with segment length but for extending tissues theinitial enhanced extension growth is independent of length;that is there is no direct linkage between the rate of extensionand auxin content. Comparisons between segments with free andsealed ends established that over 24 h some 57–73 percent of the IAA enters via the cut surfaces. Initially, thepercentage is greater; expressed as a rate per unit of surfacethe differences between cut and epidermal surfaces can reach28-fold. The rate of entry through the epidermal surface islinearly proportional to the external concentration but thisdoes not hold for cut surfaces. The addition of streptomycin,synthalin, cetyltrimethylammoniumbromide (CTAB), and chitosanto the external medium does not promote uptake of IAA by Pisumsegments; indeed synthalin is markedly inhibitory. With Gossypiumsynthalin causes little inhibition. Larger depressive effectswere induced for entry via the cut surfaces. On entry the IAAis rapidly metabolized and the rate of conversion is higherfor segments with sealed ends. These findings are discussedin relation to (a) differences in the mechanisms determiningthe uptake of IAA and other auxins, (b) cell extension and thedistribution of auxin in the tissues.     

Peroxydisulfate ion, a plant growth inhibitor, and its reaction with IAA

Germination and sprouting tests were used to demonstrate thatperoxydisulfate ion, S₂O₈^–2, reversibly inhibits turnipsprout growth but does not prevent germination. Peroxydisulfateion is kinetically inert to most organic compounds but readilyoxidizes IAA. Activation energy and rate constants for the reactionwere measured and the pH effect studied. Hormone oxidation isproposed as the effective mechanism of growth inhibition. (Received April 28, 1979; )     

Biosynthesis of Indole-3-Acetic Acid from L-Tryptophan in Coleoptile Tips of Maize (Zea mays L.)

Coleoptile tips (about 2.5 mm in length) were excised from 3-day-olddark-adapted maize (Zea mays L.) seedlings and incubated indarkness in potassium phosphate buffer that contained ¹⁴C-L-tryptophan(Trp). Subsequent analysis by gas chromatography-mass spectrometryindicated that a significant portion of endogenous indole-3-aceticacid (IAA) had been labeled with ¹⁴C. About 8% of the IAA thatdiffused from the tissue into the medium during incubation from0.5 to 2 h was labeled, and 12% of the IAA extracted from thetissue after a 2-h incubation was labeled. On the other hand,30% of the Trp extracted from the tissue after a 2-h incubationwas ¹⁴C-Trp, which was more than those determined for IAA. Sincethe experiments were carried out under the non-steady-stateconditions in which the tissue content of ¹⁴C-Trp increasedwith time, and since the extracted Trp included the ¹⁴C-Trpin the apoplastic space, it seemed that synthesis de novo fromTrp was the major means by which IAA was produced in the coleoptiletip. The conversion of Trp to IAA was not detected in sub-apicalsegments (5–7.5 mm from the top) that were incubated similarly,an indication that synthesis of IAA occurs specifically in thetip region. When intact seedlings were irradiated with a pulseof red light 2 h before excision of tips and the applicationof ¹⁴C-Trp, the amounts of extractable and diffusible IAA werereduced by 40–60% without a change in the rate of ¹⁴Cincorporation. This result indicated that the production ofIAA from Trp is controlled by a red-light signal. (Received May 15, 1995; Accepted September 1, 1995)     

Uptake and fate of IAA in apple callus tissue using IAA-1-14C

Incubation of young growing and older non-growing apple callustissues in a medium containing IAA-1-¹⁴C resulted in rapid disappearanceof the IAA. In old calluses (3 months), the major portion ofIAA was lost by decarboxylation (90% after 4 hr) and very little(1.4%) was maintained by the tissue. In young calluses, after4 hr in light, decarboxylation reached 20% and absorption 35%of the labelled IAA. Some decomposition of IAA was caused byphotolysis and autoclaving (19% and 3%, respectively) but thefinal distribution of radioactivity was not affected. Factorssuch as sucrose concentration in the incubation medium, distilledwater as incubation medium, and cutting of the callus did notaffect tissue behavior. Special precautions were taken to eliminatenon-biological decomposition of IAA. Therefore, we believe thatthe rapid CO₂ evolution is of enzymatic nature. This theoryis supported by the drop in decarboxylation after killing ofthe callus, and the increase of decarboxylation with age. Noenzyme was secreted by the callus into the medium after 24 hrof incubation, and IAA decomposition in old tissues is doneprobably on the surface. Absorption of IAA increased with increasingcallus size and decarboxylation decreased. ¹ Contribution from the Agricultural Research Organization,The Volcani Center, Bet Dagan, Israel. 1973 Series, No. 274-E. (Received May 30, 1974; )     

In vitro binding of IAA to plasma membrane-rich fractions containing Mg++-activated ATPase from mung bean hypocotyls

Cell homogenates of dark-grown mung bean hypocotyls were fractionatedinto six fractions (L-0, L-l to L-5) by stepwise sucrose density-gradientcentrifugation. The majority (ca. 84%) of Mg⁺⁺-activated ATPase activity ofthe 10,000 x g pellet was localized in the L-0 (1.03 d 1.14)and L-l (1.14 d 1.16) fractions. Over 40% of the vesicularmembrane in the L-0 fraction and 60% of the L-l fraction couldbe stained with phosphotungstic acid (PTA)-chromic acid, a selectivestaining for the plant plasma membrane. In vitro binding of ¹⁴C-IAA to the fraction components was thegreatest in the L-l fraction among the six. The binding of ¹⁴C-IAAto the L-l fraction in vitro was markedly interfered with bythe presence of a high concentration of cold IAA (2 x 10^–4M).However, it was not affected by the IAA analogues IPA, IBA andIAN. This indicates that IAA highly specifically binds to theL-l fraction. In vitro specific binding of ¹⁴C-IAA to L-l andL-0 was decreased with an increasing acidity from pH 8.0 to5.0. In vitro binding of ¹⁴C-IAA to L-l and L-5 was furtherenhanced when these fractions were isolated from sections pretreatedwith 10^–5M cold IAA for 60 min ¹Present address: Institute for Plant Virus Research, 959 Aobacho,Chiba 280, Japan. (Received August 14, 1975; )