The Uptake of Growth Substances: VII. THE ACCUMULATION OF CHLORINATED BENZOIC ACIDS BY STEM TISSUES2

The courses of uptake of benzoic acid (BA) and its 2-chloro-(2-CBA), 2,4-dichloro- (2,4-DCBA), 2,5-dichloro- (2,5-DCBA),and 2,3,6-trichloro- (2,3,6-TCBA) derivatives, all containing¹⁴C in the carboxyl group, have been investigated, employingstem segments of Pisum sativum, Gossypium hirsutum, and Avenasativa. From comparisons of the rates of accumulation by segmetns ofdifferent length it is conclueded that for each compound uptakeproceeds largely or wholly via the cut surfaces. The initial uptake of BA and 2-CBA by segments of Pisum is depressedas the pH of the solution is raised from 4 to 6.5, but the fallis less rapid than the decrease in the proportion of undissociatedmolecules. For all three species, BA and 2-CBA, which induced no extensiongrowth, were accumulated at a more or less constant rate. Bycontrast, the course of uptake of 2,3,6-TCBA, a powerful auxin,exhibited marked deviations from a linear pattern, especiallyin Avena where uptake became negative between four and six hours.This loss of radioactivity from the tissues was due to the netegress of 2,3,6-TCBA itself into the external solution. In Avenathe two dichloro-benzoic acids (2,4-DCBA and 2,5-DCBA) haveintermediate trens: net accumulation declined almost to zerobut subsequently recovered and proceeded at a rapid rate. These findings are discussed in relation to prior studies ofthe uptake of substituted phenoxyacetic acids and the conceptsof Type 1 and Type 2 accumulation. It is proposed that accumulationof BA and 2-CBA is largely governed by a stable Type 2 processwhile the initial uptake of the powerful auxins, 2,3,6-TCBAand 2,5-DCBA proceeds via an unstable system, similar or identicalto Type 1 accumulation.     

The Uptake of Growth Substances: IX. FURTHER STUDIES OF THE MECHANISM OF UPTAKE OF 2,3,6-TRICHLOROBENZOIC ACID BY AVENA SEGMENTS

In a previous paper it was established that during the courseof uptake of radioactive 2,3,6-trichlorobenzoic acid (2,3,6-TCBA)by mesocotyl segments of Avena, the rate, initially positive,became negative within six hours. This phase of net loss isprevented by streptomycin and by synthalin, while an enhancementof accumulation is brought about by cetyl trimethylammoniumbromide (CTAB). It was postulated that the initial accumulationis governed by an unstable accumulatory process (Type 1) whichinvolves adsorption by some cell membrane system through aninteraction between the carboxyl anion of the growth-regulatormolecule and the quaternary ammonium group of the choline moietyof -lecithin. Hydrolysis of lecithin by phospholipase-D destroysthis Type 1 binding, while cationic nitrogen compounds maintainpositive uptake by competing with the choline quaternary ammoniumgroup of -lecithin for the anionic site of phospholipase-D. The effects of pretreatment at a low temperature on the subsequentuptake of 2,3,6-TCBA and the influence of pH on the course ofuptake, as well as studies of the egress of choline, providesome support for the role of phospholipase-D in determiningthe instability of the accumulatory system. Synthalin and CTAB inhibit the activity of phospholipase-D invitro. However, other investigations with this enzyme alsoemphasize that such inhibition can only partially account forthe great enhancement of the uptake of TCBA produced by CTAB.Related experiments on the uptake of alkyl pyridinium compoundsby Avena segments and on their adsorption to lecithin in vitrofavour a suggestion that quaternary ammonium compounds, suchas CTAB, act largely by providing aftificial Type 1 sites. The mechanism and significance of Type 1 accumulation are discussedand compared with similar postulates for the binding of auxinsand salts.     

Degradation of mono- and dichlorobenzoic acid isomers by two natural isolates of Alcaligenes denitrificans

Two strains of Alcaligenes denitrificans, designated BRI 3010 and BRI 6011, were isolated from polychlorinated biphenyl (PCB)-contaminated soil using 2,5-dichlorobenzoic acid (2,5-DCBA) and 2,4-DCBA, respectively, as sole carbon and energy sources. Both strains degraded 2-chlorobenzoic acid (2-CBA), 2,3-DCBA, and 2,5-DCBA, and were unable to degrade 2,6-DCBA. BRI 6011 alone degraded 2,4-DCBA. Growth of BRI 6011 in yeast extract and 2,6-DCBA induced pyrocatechase activity, but 2,6-DCBA was not degraded, suggesting the importance of an unsubstituted carbon six of the aromatic ring. Metabolism of the chlorinated substrates resulted in the stoichiometric release of chloride, and degradation proceeded by intradiol cleavage of the aromatic ring. Growth of both strains on 2,5-DCBA induced pyrocatechase activities with catechol and chlorocatechols as substrates. In contrast to dichlorobenzoic acids, growth on 2-CBA, benzoic acid, mono- and dihydroxybenzoic acids induced a pyrocatechase activity against catechol only. Although 2,4-DCBA was a more potent inducer of both pyrocatechase activities, its utilization by BRI 6011 was inhibited by 2,5-DCBA. Specific uptake rates using resting cells were highest with 2-CBA, except when the resting cells had been previously grown on 2,5-DCBA, in which case 2,5-DCBA was the preferred substrate. The higher rates of 2,5-DCBA uptake obtained by growth on that substrate, suggested the existence of a separately induced uptake system for 2,5-DCBA.     

The Uptake of Growth Substances: V. VARIATION IN THE UPTAKE OF A SERIES OF CHLORINATED PHENOXYACETIC ACIDS BY STEM TISSUES4GOSSYPIUM HIRSUTUM AND ITS RELATIONSHIP TO DIFFERENCES IN AUXIN ACTIVITY

When segments excised from the etiolated hypocotyls of Gossypiumhirsutum are pretreated in buffer, the subsequent uptake ofradioactive 2,4-dichlorophenoxyacetic acid (2,4-D-1-¹⁴C) isdepressed and the net loss of radioactivity which normally followsa phase of positive uptake by freshly excised segments doesnot take place. Uptake by fresh segments, in contrast with uptakeafter pretreatment, has a high Q₁₀ and is markedly depressedby both 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and 3-indolylaceticacid. On these grounds it is proposed that net loss resultsfrom the release of material accumulated by a specific mechanismwhich, with time, becomes inoperative. Additional experimentssuggest that part of the 2,4-D taken up by stem segments ofTriticum vulgare and Avena sativa is accumulated by a similarmechanism. For 1-cm segments, entry is most rapid through the cut ends,and the effects of pretreatment exert their maximal effectsin the tissue near the ends. Therefore very short segments havebeen used to compare the courses of uptake of phenoxyaceticacid (POA) and its 2-, 4-, 2,6-, 2,4- and 2,4,5- chloro- derivatives.The patterns observed are similar to those previously reportedfor 1 -cm segments, although the differences between compoundsare greater. The courses of uptake of 2,4-D and 2,4,5-T, bothterminate in a phase when there is a net loss. POA and the 2-chloro-substitutedacid (2-CPA) are both continuously accumulated. No net lossis found with either the 2,6- (2,6-D) or the 4- chloro (4-CPA)compounds but the rates of uptake progressively decrease toa low level. It is proposed that the processes which determine the patternof uptake of chlorinated phenoxyacetic acids include two typesof accumulation. With Type I accumulation the mechanisms involvedrapidly become disorganized after tissues are excised from theplant. Type 2 accumulation, on the other hand, is stable. Theavailable data indicate that Type I accumulation is peculiarto compounds with marked auxin-like properties.     

The Uptake of Growth Substances: XIV. PATTERNS OF UPTAKE BY LEMNA MINOR OF PHENOXYACETIC AND BENZOIC ACIDS FOLLOWING PROGRESSIVE CHLORINATION

The interrelationships between chemical structure and patternsof uptake by Lemna minor have been examined for (a) phenoxyaceticacid and its 2-, 4-, 2,4-, 2,6-, 3,5-, 2,5-, 2, 4, 5- and 2,4,6-chloroderivatives and (b) benzoic acid and its 2-, 2,4-, 2,5-, 2,3,6-chloro-and 3,6-dichloro-2-methoxy derivatives. All compounds contained¹⁴C in the carboxyl group. The plants from a clonal populationwere grown at a constant temperature and continuously illuminated. With progressive chlorination of phenoxyacetic acid, uptakeis enhanced, so that by 6 h there is a fourfold difference betweenthe monochloro- and trichloro-derivatives. In complete contrast,chlorination of benzoic acid greatly suppresses uptake and thedifferences associated with the degree of chlorination are smaller. Arising out of previous studies, the effects of adding streptomycin,synthalin, and cetyltri-methylammoniumbromide on the courseof uptake of individual members of the two series have beenexamined. Each of the additions can induce positive, negative,or null changes in the pattern of uptake, but the nature ofthe response is also dependent on the properties of the compound. These findings are discussed in relation to prior studies concerning(a) penetration into the leaves of Phaseolus vulgaris, (b) uptakeby excised segments of etiolated stems, and (c) changes in physico-chemicalproperties resulting from progressive chlorination. Many of the complexities still remain to be resolved but itseems clear that adsorption by Borne membrane system involvingthe carboxyl group of the entering acid and the positively chargedquaternary ammonium group of alpha-lecithin cannot be restrictedto compounds which are physiologically active as auxins or herbicides.     

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.     

The Uptake of Growth Substances: X. THE ACCUMULATION OF PHENOXYACETIC ACID AND 2,4-DICHLOROPHENOXYACETIC ACID BY SEGMENTS OF AVENA MESOCOTYL

Segments of Avena mesocotyl were placed in buffered solutionsof phenoxyacetic acid (POA) or 2,4-dichlorophebnoxyacetic acid(2,4-D), containing carbon-14 in the carboxyl group, and thequantities of radioactivity taken up by the tissues measured.With freshly cut segments in solutions of 2,4-D there is accumulationof carbon-14, but the course of uptake is interrupted by a temporaryphase when some of the accumulated 2,4-D is released to theexternal solution. If after cutting the segments are first pretreatedby placing them for about 15 h in buffer, and then transferredto 2,4-D, there is progressive accumulation with no phase ofnet loss. Pretreated segments absorb greater quantities of either 2,4-Dor POA than freshly excised tissues. Following pretreatmentin buffer the course of uptake of POA is linear but for 2,4-Dthe course is curvilinear. However, after pretreatment withnon-radioactive 2,4-D the subsequent rate of uptake of radioactive2,4-D is constant over long periods. The uptake of radioactive2,4-D is largely independent of the concentration of non-radioactive2,4-D given during pretreatment. When segments which have absorbed 2,4-D-1-¹⁴C are transferredto buffer, a relatively small proportion of the carbon-14, the‘mobile fraction’, is released. The amount releasedfollowing different periods of uptake is constant whereas thelevel of non-mobile carbon-14, the ‘residual fraction’,rises progressively in step with accumulation. The uptake of POA and 2,4-D is accompained by the formationwithin the tissues of other radioactive substances. It is concludedthat the residual fraction is composed, at least in part, ofthese metabolic products and that accumulation and metabolicconversion are inter-connected. Dinitrophenol (DNP) slowly and progressively depresses the uptakeof POA whereas the uptake of 2,4-D is very rapidly arrested.However, after about 2 h, in the continued presence of DNP,uptake of 2,4-D restarts but the rate never attains that ofthe control. These divergent effects of DNP indicate that POAand 2,4-D are accumulated by different pathways.     

The Uptake of Growth Substances: XV. THE DIFFERENTIAL EFFECTS OF PROGRESSIVE CHLORINATION OF PHENOXYACETIC ACID ON ENTRY INTO THE EPIDERMAL AND CUT SURFACES OF STEM SEGMENTS

The comparative patterns of entry into segments with sealedand open ends, excised from etiolated internodes of Pisum sativum,have been examined for phenoxyacetic acid (POA) and its 2-,4-, 2,4-, 2,6-, 3,5-, 2,4,5- and 2,4, 6-chloro derivatives,each containing ¹⁴C in the carboxyl group. Sealing the ends greatly depresses the level of entry, on averagean eight-fold reduction at 9 h. Likewise, the interrelationsbetween the degree of chlorination and uptake potential aredisparate. For segments with exposed cut surfaces the finalcontent is maximal for POA and the 2-chloro compound and minimalfor the 3,5-dichloro derivative (3,5-D) with an eight-fold difference.With sealed ends this difference is reduced to two-fold butwhile 3,5-D accumulates least uptake is now highest for POAand 2,4-D. There are also changes in the order with time. Initially,2,4,5-T penetrates fastest into sealed segments but for segmentswith open ends entry is most rapid for the 4- and 2,4,6-chloroderivatives. Additions of streptomycin and cetyltrimethylammoniumbromide(CTAB) induce differential changes in the patterns of uptake.Where uptake is promoted the enhancement is not restricted toactive auxins. Sealing the ends may alter the nature of theresponse. The likely physico-chemical and metabolic processes concernedin the two routes of entry are discussed and the results comparedwith previous divergent findings on the relationship betweenchemical structure and uptake by Lemna minor and penetrationinto leaves of Phaseolus vulgaris.     

The Uptake of Growth Substances: VI. A COMPARATIVE STUDY OF THE FACTORS DETERMINING THE PATTERNS OF UPTAKE OF PHENOXYACETIC ACID AND 2,4,5-TRICHOLOROPHEN-OXYACETIC ACID, WEAK AND STRONG AUXINS, BY GOSSYPIUM TISSUES2

Using segments of etiolated hypocotyls of Gossypium, a comparativestudy has been made of the processes which determine the patternsof uptake of a very weak auxin, phenoxyacetic acid (POA), anda very powerful one, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). When segments are placed in solutions of POA-1-¹⁴C, a continuousincrease in the radioactivity of the tissues is accompaniedby the formation of radioactive metabolities which can be separatedfrom POA by techniques of paper chromatography. At the sametime there is a progressive increase in the amount of radio-activitywhich cannot be removed by transferring the tissues to buffer.Uptake is inhibited by low temperature, anaerobiosis, 2,4-dinitrophenol,and iodoacetate. It is concluded that the accumulation of POAinvolves its metabolic conversion to products which do not readilydiffuse out into the external medium. With 2,4,5-T-1-¹⁴C the radioactivity of the segments at firstincreases rapidly but this is followed after two hours by aphase of rapid decrease. No radioactive metoabolites can bedetected by paper chromatography and all of the ¹⁴C taken upcan be rapidly removed by transfer to buffer. The magnitudeof the decrease in radioctivity during the second phase of uptakeis balanced by a release to the medium of a matched amount ofradioactive 2,4,5-T. Uptake of 2,4,5-T is somewhat less sensitiveto temperature and anaerobiosis than uptake of POA and is bycontrast only slightly inhibited by 2,4-dinitrophenol and iodoacetate. Pretreatment of segments in buffer markedly alters the patternof uptake of 2,4,5-T but not that of POA. It reduces both theamount of 2,4,5-T initially taken up and the amount subsequentlyreleased to the medium. In addition, both net loss of radioactivityduring the course of uptake of 2,4,5-T and the reduction inthe extent of uptake following pretreatment are both arrestedby adding streptomycin, but not by the addition of pencillinor chloramphenicol. It is concluded that the uptake of 2,4,5-T involves reversibleaccumulation by a process whose efficiency decreases with time:the most likely systems are a metabolically linked mechanismfor the active transport across a membrane or reversible adsorptionon specific binding sites.     

The Uptake of Growth Substances: II. THE ABSORPTION AND ACCUMULATION OF 2:3:5-TRIIODOBENZOIC ACID BY THE ROOT AND FROND OF LEMNA MINOR

The absorption by Lemna minor of 2 : 3 : 5-triiodobenzoic acid(TIBA), labelled in the 2 position with iodine-131, has beeninvestigated. Under constant conditions of light (300 foot candles)and temperature (25?C.) the course of uptake by the whole plantis dependent on the concentration of TIBA in the culture solution(pH 5.I). Up to circa 1 mg./l. a high rate of uptake in thefirst 30 minutes is followed by a slower but steady accumulationduring the next 4.5 hours. When the concentration is increasedto 5 mg./1. uptake, at first rapid, falls off progressivelyso that after 1-2 hours the content of the tissues starts todecrease because TIBA moves out in to the external solutionfaster than it enters. The magnitude of this reversal from apositive to a negative rate of uptake,.partitularly for theroots, is even more pronounced at 10 mg/l. When plants, first placed in labelled solutions for 2 to 2.5hours are transferred to culture solution the loss of the labelledcompound to the external solution, especially from the roots,is very rapid : the decrease in root content may reach 90 percent. in the first 80 minutes. The initial rate of egress islittle affected either by the hydrogen ion concentration orthe presence of unlabelled TIBA in the external solution. Between5, 15, and 25? C. the temperature quotients range from 1.29to 1.70 for the root and 1.66-1.37 for the frond. Comparable experiments on the initial phase of uptake duringthe first 2o to 30 minutes demonstrate that (a) the rates aregreatly dependent on the external pH i.e. uptake is largelyin the molecular form and (b) between 5 and 15?C. the ratesare not temperature dependent but between 15 and 25?C. theyare: the quotients for roots and frond are 2.6 and 2.0. Comparisons of the course of uptake of normal plants and plantspreviously frozen in liquid air showed that at both I? and 25?C.'pre-frozen' fronds continue to accumulate TIBA and may containat the end of 5 hours 8 times as much as control fronds. Incontrast, the uptake by the pre-frozen roots is depressed. Theaddition of high (30-50 mg./l.) but not low (10 mg./1.) concentrationsof indole-3-acetic acid (IAA) to the external solution alsocauses a striking accumulation of TIBA in the frond but uptakeby the roots is not changed. Lastly, the presence of TIBA in the external solution whilenot affecting the uptake of cerium-144. in the first 2 hourssubsequently depresses uptake by the root but not by the frond. These findings are compared with the similar pattern of uptakefor z,4-dichlorophenoxyacetic acid described in the previouspaper. The nature of the processes controlling the inward andoutward flow of TIBA, their location within the cell and theirbearing on the physiological actions of auxins are discussed.     

The Uptake of Growth Substances: XI. VARIATIONS IN THE ACCUMULATION OF SUBSTITUTED PHENOXYACETIC ACIDS OF DIFFERING PHYSIOLOGICAL ACTIVITY BY SEGMENTS OF AVENA MESOCOTYL

An examination has been made of the phase of continuous accumulationof phenoxyacetic acid (POA) and the 2-, 4-, 2,4-, 2,6-, and2,4,5-chloro-derivatives, containing carbon-14 in the carboxylgroup, by segments of the Avena mesocotyl. On the basis of previousfindings to eliminate the initial transient components of uptake(Type I processes) the segments were pretreated for 13 to 18h in buffer or buffer containing the respective non-radioactivecompound. For five of the compounds the relationship between the rateof uptake and the external concentration takes the form of arectangular hyperbola, but for the sixth, 2,4,5-T, this relationshipdoes not hold. The data, except those for 2,4,5-T, have beenevaluated as linear regressions of rate of uptake against thequotient of rate over concentration. From each regression equationtwo constants have been derived: the point ‘B’ wherethe line intersects the rate axis (the theoretically maximumrate) and the slope of the regression ‘K’, whichcan alternatively be expressed as the concentration at whichthe observed rate equals half the value of ‘B’. The calculated values of B and K for POA are approximately twiceas great as the corresponding values for 2-CPA, and about 25times greater than for 4-CPA. The values for 2,4-D are closeto those for 4-CPA, and 2,6-D is intermediate between 2-CPAand 4-CPA. Although the constants for 2,4,5-T could not be calculatedprecisely, the rates of accumulation are about one-fourth ofthose measured for 2,4-D at equivalent concentrations. The uptake of radioactive 2,4-D is slightly depressed in thepresence of nonradioactive POA. Greater reductions are causedby 2-CPA or 2,6-D, and 2,4-D or 2,4,5-T are even more inhibitory.The pattern of inhibition caused by 2,4,5-T indicates competitionfor common sites of uptake, while POA appears not to be competitive.In corresponding experiments with POA, the presence of the otherregulators only caused small inhibitions and the order was different. Earlier work showed that in Avena accumulation is accompaniedby the conversion to a varying degree of the individual substitutedphenoxyacetic acids to conjugated derivatives. It is suggestedthat the variation between compounds in their rates of accumulationis in part due to differences in the stability of the conjugatedderivatives, and that the facility of conversion is a factorin determining physiological activity.     

The effects of 2,4-dinitrophenol and chemical modifying reagents on auxin transport by suspension-cultured crown gall cells

1. The effects of 2,4-dinitrophenol (DNP) and chemical modifying reagents on the transport of indol-3-yl acetic acid (IAA) and 2,4-dichlorophenoxyacetic acid (2,4 D) by suspension-cultured crown gall cells of Parthenocissus tricuspidata Planch. were investigated. 2. DNP smoothly reduced uptakes of both benzoic acid and 2,4 D but IAA uptake at pH 6.0 was not inhibited by concentrations below 20 mol/l except in the presence of 2,3,5-triiodobenzoic acid (TIBA) whose stimulatory effect was thereby abolished. DNP stimulated the efflux of 2,4 D and of IAA in the presence of TIBA. Without TIBA, DNP first inhibited but later stimulated IAA efflux. —3. Low concentrations of N-ethylmaleimide (NEM) (<5 mol/l) abolished TIBA-stimulation of net IAA uptake while not affecting (or slightly promoting) net uptake of IAA alone, whose inhibition needs greater NEM concentrations. Diethylpyrocarbonate behaved similarly. The poorly-penetrant p-chloromercuriben-zenesulphonic acid did not cause a marked differential inhibition of the TIBA stimulation. — 4. Together with earlier data, the results support a two-carrier model comprising a common carrier for IAA and 2,4 D, previously suggested to be an auxin anion/proton symport, and also an electrogenic carrier, specific for IAA anions, and inhibited by TIBA. The role of such carriers in polar auxin transport is discussed.Abbreviations IAA Indol-3-yl acetic acid - 2,4 D 2,4-Dichlorophenoxyacetic acid - BA Benzoic acid - DNP 2,4-Dinitrophenol - NEM N-ethylmaleimide - PCMBS p-Chloromercuribenzenesulphonic acid - TIBA 2,3,5-Triiodobenzoic acid     

Components of auxin transport in stem segments of Pisum sativum L.

1. The uptake of indol-3-yl acetic acid ([1-¹⁴C]IAA, 0–2.0 M) into light-grown pea stem segments was measured under various conditions to investigate the extent to which mechanisms of auxin transport in crown gall suspension culture cells (Rubery and Sheldrake, Planta 118, 101–121, 1974) are also found in a tissue capable of polar auxin transport. — 2. IAA uptake increased as the external pH was lowered. IAA uptake was less than that of benzoic acid (BA), naphthylacetic acid (NAA) or 2,4 dichlorophenoxyacetic acid (2,4D) under equivalent conditions. TIBA enhanced net IAA uptake through inhibition of efflux, and to a lesser extent, also increased uptake of NAA and 2,4D while it had no effect on BA uptake. — 3. Both DNP and, at higher concentrations, BA, reduced IAA uptake probably because of a reduction of cytoplasmic pH. However, low concentrations of both BA and DNP caused a slight enhancement of IAA net uptake, possibly through a reduction of carrier-mediated IAA efflux. In the presence of TIBA, the inhibitory effects of DNP and BA were more severe and there was no enhancement of uptake at low concentrations. — 4. Non-radioactive IAA (10 M) reduced uptake of labelled IAA but further increases in concentration up to 1.0 mM produced first an inhibition (0–10 min) of labelled IAA uptake, followed by a stimulation at later times. Non-radioactive 2,4 D decreased, but was not observed to stimulate, uptake of labelled IAA. In the presence of TIBA labelled IAA uptake was inhibited by non-radioactive IAA regardless of its concentration. — 5. Sulphydryl reagents PCMB and PCMBS promoted or inhibited IAA uptake depending, respectively, on whether they penetrated or were excluded from the cells. The penetrant PCMB also reduced the promotion of labelled IAA uptake by TIBA or by high concentrations of added non-labelled IAA. — 6. Our findings are interpreted as being consistent with the diffusive entry of unionised IAA into cells together with some carrier-mediated uptake. Auxin efflux from the cells also appears to have a carrier-mediated contribution, at least part of which is inhibited by TIBA, and which has a capacity at least as great as that of the uptake carrier. The data indicate that pea stem segments contain cells whose mechanisms of trans-membrane auxin transport fit the model of polar auxin transport proposed from experiments with crown gall suspension cells, although differences, particularly of carrier specificity, are apparent between the two systems.Abbreviations IAA indol-3-yl acetic acid - BA benzoic acid - NAA 1-naphthylacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - TIBA 2,3,5-triiodobenzoic acid - DNP 2,4-dinitrophenol - PCMB p-chloromercuribenzoic acid - PCMBS p-chloromercuribenzene sulphonic acid This work was performed in Cambridge during the tenure of a sabbatical leave by P.J.D. Supported by a grant for supplies from the American Philosophical Society to P.J.D.     

The role of auxin efflux carriers in the reversible loss of polar auxin transport in the pea (Pisum sativum L.) stem

Correlatively inhibited pea shoots (Pisum sativum L.) did not transport apically applied ¹⁴C-labelled indol-3yl-acetic acid ([¹⁴C]IAA), and polar IAA transport did not occur in internodal segments cut from these shoots. Polar transport in shoots and segments recovered within 24 h of removing the dominant shoot apex. Decapitation of growing shoots also resulted in the loss of polar transport in segments from internodes subtending the apex. This loss was prevented by apical applications of unlabelled IAA, or by low temperatures (approx. 2° C) after decapitation. Rates of net uptake of [¹⁴C]IAA by 2-mm segments cut from subordinate or decapitated shoots were the same as those in segments cut from dominant or growing shoots. In both cases net uptake was stimulated to the same extent by competing unlabelled IAA and by N-1-naphthylphthalamic acid. Uptake of the pH probe [¹⁴C]-5,5-dimethyloxazolidine-2,4-dione from unbuffered solutions was the same in segments from both types of shoot. Patterns of [¹⁴C]IAA metabolism in shoots in which polar transport had ceased were the same as those in shoots capable of polar transport. The reversible loss of polar IAA transport in these systems, therefore, was not the result of loss or inactivation of specific IAA efflux carriers, loss of ability of cells to maintain transmembrane pH gradients, or the result of a change in IAA metabolism. Furthermore, in tissues incapable of polar transport, no evidence was found for the occurrence of inhibitors of IAA uptake or efflux. Evidence is cited to support the possibility that the reversible loss of polar auxin transport is the result of a gradual randomization of effluxcarrier distribution in the plasma membrane following withdrawal of an apical auxin supply and that the recovery of polar transport involves reestablishment of effluxcarrier asymmetry under the influence of vectorial gradients in auxin concentration.Abbreviations DMO 5,5-dimethyloxazolidine-2,4-dione - IAA indol-3yl-acetic acid - NPA N-1-naphthylphthalamic acid - TIBA 2,3,5-triiodobenzoic acid This work was supported by grant no. GR/D/08760 from the U.K. Science and Engineering Research Council. We thank Mrs. R.P. Bell for technical assistance.     

Studies on Foliar Penetration: IV. MECHANISMS CONTROLLING THE RATE OF PENETRATION OF 2,4-DICHLOROPHENOXYACETIC ACID (2,4-D) INTO LEAVES OF PHASEOLUS VULGARIS

The effects of a wide range of metabolic inhibitors on the penetrationof 2,4-dichlorophenoxyacetic acid (2,4-D) into the leaf disksof Phaseolus vulgaris have been studied. While recognizing thelack of specificity of most inhibitors, compounds were chosenwhich are known to affect respiration, phosphorylation, photosynthesis,membrane permeability, protein synthesis, and the binding capacityof membrane systems. They were: fluoride, azide, arsenite, iodoacetate,arsenate, 2,4-dinitrophenol (DNP), 3-(3,4-dichlorophenyl), -I,I-dimethylurea (DCMU), phenylmercuric chloride, octenylsuccinicacid, decenylsuccinic acid, dimethyl sulphoxide, actinomycin-D,chloramphenicol, streptomycin, 5-fluorouracil, cycloheximide,and cetyltrimethylammoniumbromide (CTAB).At sub-toxic levelsall compounds had little or no influence on penetration in darknesssave for iodoacetate and decenylsuccinic acid, which causedsome enhanced entry at 10^-4M and 10^-3M respectively, and CTABwhich promoted penetration at concentrations known tolower thesurface tension of water.The much greater rate of penetrationof 2,4-D into disks exposed to bright light (16 000 lx) is unaffectedby fluoride, azide, DNP, octenylsuccinic acid, decenylsuccinicacid, dimethyl sulphoxide, or actinomycin-D. It is, however,progressively inhibited by increasing concentrations of arsenite,iodoacetate, arsenate, streptomycin, and 5-fluorouracil. Chloramphenicol,cycloheximide, and CTAB lower the rate of penetration at intermediateconcentrations but at high concentrations the affect is reversed.The most active inhibitors of light-induced penetration areDCMU and phenylmercuric chloride, compounds which block theproduction of ATP.These results are discussed in relation tomechanisms of transport, in particular the structureand stabilityof barriers likely to impede penetration.     

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.     

The specificity of carrier-mediated auxin transport by suspension-cultured crown gall cells

1. The specificity of the auxin transport system of suspension-cultured crown gall cells from Parthenocissus tricuspidata Planch- is examined with regard to 2,4-Dichlorophenoxyacetic acid (2,4 D), l-Naphthylacetic acid (NAA) and Benzoic acid (BA) as well as for indole-3-acetic acid (IAA). — 2. All four weak acids can be accumulated by the cells from a medium more acidic than the cytoplasm. This is by virtue of non-specific passive diffusion of their lipid-soluble protonated forms down a concentration gradient. The corresponding anionic species are much less permeant. The extent of the accumulation is dependent on the pH difference that is maintained by the cells between their cytoplasm and the incubation medium. Studies of the concentration dependence of BA and NAA net uptake at a series of external pHs suggest that an acidification of the cytoplasm can be eventually brought about by the entry of weak acid into the cells. — 3. The uptake of 2,4 D, as well as that of IAA, has a saturable carrier-mediated component in addition to the passive diffusion of the undissociated acid. These saturable components probably represent anion uptake and appear to be mediated by a common carrier. The kinetic studies provided no evidence for the participation of carriers in the transport of BA or NAA. — 4. It was shown that the efflux of 2,4 D also has a carrier-mediated component and it is suggested that both the influx and efflux of IAA and 2,4 D occur on a common carrier. — 5. The inhibitor of polar auxin transport, 2,3,5-triiodobenzoic acid (TIBA), stimulates the net uptake of IAA by inhibiting carrier-mediated efflux of IAA from the cells. However, TIBA could not be demonstrated to have a significant effect on 2,4 D transport and any perturbation that occurs is very small in comparison with its effect on IAA movement. To account for this, the proposed common carrier could exhibit some difference in its internal binding characteristics betweend 2,4 D and IAA. An alternative explanation is that a second carrier is present, which mediates IAA efflux only, and which is inhibited by TIBA. — 6. TIBA has no significant effect on the transport of either BA or NAA, except to bring about an inhibition of net uptake, and a corresponding stimulation of efflux, when it is present at concentrations sufficient to acidify the cytoplasm. —7. The crown gall cells are compared to intact plant tissues capable of polar auxin transport with regard to the specificities exhibited for the transport of the auxins IAA, 2,4 D and NAA and the non-auxin BA.Abbreviations IAA indol-3-yl acetic acid - 2,4 D 2,4-Dichlorophenoxyacetic acid - NAA 1-Naphthylacetic acid - BA Benzoic acid - TIBA 2,3,5-triiodobenzoic acid     

Growth Substance Interactions during Uptake by Coleoptile Segments of Avena sativa and Hypocoty1 Segments of Phaseolus radiatus

Further studies have been made on the interactions of plant-growthregulators during uptake by Avena sativa coleoptile and Phaseolusradiatus hypocotyl segments. 2, 4-Dichlorophenoxyacetic acid(2, 4-D) had no effect on the uptake of either indol-3yl-aceticacid (IAA) or -naphthylacetic acid (NAA) by Avena. On the otherhand, a-(i-naphthylmethylthio)-propionic acid (NMSP) stronglyinhibited IAA uptake non-competitively but was much less effectiveon NAA uptake by Avena. The ‘metabolic’ uptake ofIAA by hypocotyl segments of Phaseolus radiatus was very stronglyinhibited by 2, 3, 5-tri-iodobenzoic acid (TIBA).     

Uptake of Benzoic Acid and Chloro-Substituted Benzoic Acids by Alcaligenes denitrificans BRI 3010 and BRI 6011

The mechanism of uptake of benzoic and 2,4-dichlorobenzoic acid (2,4-DCBA) by Alcaligenes denitrificans BRI 3010 and BRI 6011 and Pseudomonas sp. strain B13, three organisms capable of degrading various isomers of chlorinated benzoic acids, was investigated. In all three organisms, uptake of benzoic acid was inducible. For benzoic acid uptake into BRI 3010, monophasic saturation kinetics with apparent K(infm) and V(infmax) values of 1.4 (mu)M and 3.2 nmol/min/mg of cell dry weight, respectively, were obtained. For BRI 6011, biphasic saturation kinetics were observed, suggesting the presence of two uptake systems for benzoic acid with distinct K(infm) (0.72 and 5.3 (mu)M) and V(infmax) (3.3 and 4.6 nmol/min/mg of cell dry weight) values. BRI 3010 and BRI 6011 accumulated benzoic acid against a concentration gradient by a factor of 8 and 10, respectively. A wide range of structural analogs, at 50-fold excess concentrations, inhibited benzoic acid uptake by BRI 3010 and BRI 6011, whereas with B13, only 3-chlorobenzoic acid was an effective inhibitor. For BRI 3010 and BRI 6011, the inhibition by the structural analogs was not of a competitive nature. Uptake of benzoic acid by BRI 3010 and BRI 6011 was inhibited by KCN, by the protonophore 3,5,3(prm1), 4(prm1)-tetrachlorosalicylanilide (TCS), and, for BRI 6011, by anaerobiosis unless nitrate was present, thus indicating that energy was required for the uptake process. Uptake of 2,4-DCBA by BRI 6011 was constitutive and saturation uptake kinetics were not observed. Uptake of 2,4-DCBA by BRI 6011 was inhibited by KCN, TCS, and anaerobiosis even if nitrate was present, but the compound was not accumulated intracellularly against a concentration gradient. Uptake of 2,4-DCBA by BRI 6011 appears to occur by passive diffusion into the cell down its concentration gradient, which is maintained by the intracellular metabolism of the compound. This process could play an important role in the degradation of xenobiotic compounds by microorganisms.     

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.