The Uptake of Growth Substances: VIII. ACCUMULATION OF CHLORINATED BENZOIC ACIDS BY AVENA SEGMENTS: A POSSIBLE MECHANISM FOR THE TRANSIENT PHASE OF ACCUMULATION

If segments from the mesocotyls of Avena sativa are first keptin buffer then the initial rates of uptake of radioactive 2,3,6-trichlorobenzoicacid (2,3,6-TCBA) and 2,4- and 2,5-dichlorobenzoic acids (2,4-DCBAand 2,5-DCBA) are less than those of freshly excised segments.No such effect of pretreatment is found for benzoic acid orfor 2-chlorobenzoic acid (2-CBA). Uptake of 2,3,6-TCBA normallybecomes negative between two and six hours after excision, andthis phase of net loss is prevented by the addition of streptomycin,which also offsets the decline in the rates of uptake of 2,5-DCBAand 2,4-DCBA. In contrast, streptomycin inhibits accumulationof 2-CBA. From a comparison of these results with similar andprior findings for substituted phenoxyacetic acids, it is concludedthat the initial uptake of 2,3,6-TCBA, 2,5-DCBA, and 2,4-DCBAis governed by an unstable accumulatory system (Type 1), whosebreakdown can result either in a phase of net loss during thecourse of uptake, or in a decline in uptake following pretreatment. Net loss of 2,3,6-TCBA is also prevented by synthalin (decamethylenediguanidine dihydrochloride), cetyl trimethyl ammonium bromide(CTAB) and by 2,3,5-triiodobenzoic acid (TIBA). During pretreatment,the presence of streptomycin, synthalin or TIBA prevents a fallin the subsequent uptake of 2,3,6-TCBA, while the addition ofCTAB causes a dramatic increase in uptake. We have proposed for Type 1 accumulation a biochemical mechanismcapable of accounting for the unstable nature of the accumulationand for the protective action of the compounds with cationicnitrogen groups, such as streptomycin, synthalin, and CTAB.     

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: 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: 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: 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: 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.     

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).     

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.     

Inhibitory effect of lycorine on cell division and cell elongation

Lycorine, an alkaloid isolated from bulbs of Amarillidaceae,was found to be a powerful inhibitor of cell division and elongation.Adding different concentrations of lycorine from 10^–6M to 10^–4 M in an appropriate growth-medium strongly inhibitedcell division in explants of lettuce pith parenchyma. The sameresult was obtained with liquid yeast cultures growing exponentially. Lycorine-treated meristematic cells of the primary roots ofVicia faba also showed rapid inhibition of the mitotic indexwhile interphase cells increased proportionately. Lycorine alsoinhibited endogenous and auxin-induced cell elongation in Avenacoleoptiles and pea segments. Since both cell division and cell elongation require proteinsynthesis and RNA synthesis, the assumption is that lycorineprobably inhibits one of the two syntheses. ¹This study was supported by a contract between the NationalResearch Council of Italy and University of Bari, Instituteof Botany. (Received November 27, 1972; )     

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.     

Induction of the halobenzoate catabolic pathway and cometabolism of ortho-chlorobenzoates in Pseudomonas aeruginosa 142 grown on glucose-supplemented media

The aerobic cometabolism of ortho-substitutedchlorobenzoates by Pseudomonas aeruginosa strain 142 growing on glucose-supplemented medium was analyzed. The strain, which can use 2-chlorobenzoate (2-CBA) and 2,4-dichlorobenzoate (2,4-DCBA) as sole carbon and energy sources, showed high rates of 2-CBA metabolism in glucose-fed cells. In contrast, 2,4-DCBA was metabolized only after extended incubation of the full grown culture and depletion of glucose.In addition to the ortho-dehalogenation (ohb ₁₄₂) genes encoding the and subunits of the oxygenase component of a 2-halobenzoate dioxygenase, strain 142 harbours a closely relatedohbABCDFG gene cluster previously identified inP. aeruginosa JB2 (ohb _JB2). The genes for the chlorocatechol ortho-catabolic pathway were identified andsequenced in this strain, showing a near complete identity with the clcABD operon of the pAC27 plasmid. Relative quantification of mRNA by RT-PCR shows apreferential induction of ohb ₁₄₂ by 2-CBA, which is abolished in glucose-grown cultures. The alternate ohb _JB2and clc genes were expressed preferentially in 2,4-DCBAgrown cultures. Only ohb _JB2appears to be expressed in the presence of the carbohydrate. Detection of chlorocatechol-1,2-dioxygenase activity in 2,4-DCBA plus glucose grown cultures suggests the presence of an alternate system for the ortho-cleavage of chlorobenzoates. The recruitment of elements from two halobenzoate dioxygenase systems with different induction patterns, together with achlorocatechol degradative pathway not repressed by carbon catabolite, may allow P. aeruginosa 142 to cometabolize haloaromatics in carbohydrate grown cultures.     

Influence of Several Growth Regulators and Amino Acids on in vitro Organogenesis of Torenia fournieri Lind.

The effects of several growth regulators and amino acids onin vitro organogenesis of Torenia fournieri Lind. were determinedusing internodal segments. Treatment with 2,4-D¹ resulted innodular callus formation, while NAA and IAA induced roots constantlybut much less frequently shoot buds. Individually BA, zeatin,and 4-PU induced bud formation, but these shoot buds did notdevelop further. Formation of buds by cytokinin was influencedby a simultaneous application of NAA or 2,4-D, but not of IAA,its degree being reduced when BA was simultaneously appliedwith NAA or 2,4-D. When zeatin or kinetin was added with NAA,numerous roots were induced. The effects of various L-amino acids on in vitro organogenesiswere also investigated using the defined medium in which KNO₃was a principal source of nitrogen. The formation of buds wasconsiderably stimulated by alanine and asparagine, and slightlyby glutamic acid in the medium containing both NAA and BA, inwhich bud formation was easily induced. On the other hand, allamino acids except for glutamic acid and aspartic acid inhibitedroom formation in this medium. Root formation was greatly stimulated by proline, alanine, glutamine,glutamic acid, and aspartic acid, and slightly by arginine andtryptophan in the medium containing NAA but no BA. Glutamicacid and aspartic acid also enhanced bud formation in this medium.     

Effects of Inhibitors of Protein Synthesis on Transmembrane Auxin Transport in Cucurbita pepo L. Hypocotyl Segments

Pretreatment of 2?0 mm segments of etiolated zucchini (Cucurbitapepo L.) hypocotyl with cycloheximide (CH) or 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide(MDMP) eliminated the stimulation by N-1-naphthylphthalamicacid (NPA) of net uptake of [1-¹⁴C]indol-3yl-acetic acid ([1-¹⁴C]IAA),but had relatively little effect on the net uptake of IAA inthe absence of NPA. The efflux of [1-¹⁴C]IAA from preloadedsegments was not substantially affected by inhibitor pretreatmentin the absence of NPA, but CH pretreatment significantly inhibitedthe reduction of efflux caused by NPA. Pretreatment with CHor MDMP did not affect net uptake by segments of the pH probe[2-¹⁴C]5,5-dimethyl-oxazolidine-2,4-dione ([2-¹⁴C]DMO), or thenet uptake of [¹⁴C]-labelled 3-O-methylglucose ([¹⁴C]3-0-MeGlu),suggesting that neither inhibitor affected intracellular pHor the general function of proton symporters in the plasma membrane.Both compounds reduced the incorporation of label from [³⁵S]methionineinto trichloroacetic acid (TCA)-insoluble fractions of zucchinitissue, confirming their inhibitory effect on protein synthesis. The steady-state association of [³H]IAA with microsomal vesiclesprepared from zucchini hypocotyl tissue was enhanced by theinclusion of NPA in the uptake medium. The stimulation by NPAof [³H]IAA association with microsomes was substantially reducedwhen the tissue was pretreated with CH. However, CH pretreatmentdid not affect the level of high affinity NPA binding to themembranes indicating that treatments did not result in lossof NPA receptors. It is suggested that the auxin transport site on the effluxcarrier system and the receptor site for NPA may reside on separateproteins linked by a third, rapidly turned-over, transducingprotein. Key words: Auxin carriers, auxin efflux, Cucurbita pepo, phytotropin receptors     

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.     

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

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

Effect of kinetin on auxin uptake and distribution in normal and tumor-prone Nicotiana shoots

Stem segments of non-tumorous Nicotiana glauca and N. langsdorffiiplants and of their tumor-producing amphidiploid F₁ hybrid weretreated with 6-furfurylaminopurine (kinetin) prior to transporttests with applied labeled indoleacetic acid (IAA-2-¹⁴C). Kinetin-treatmentsincreased the uptake of IAA in non-tumorous shoots; the IAAuptake by N. langsdorffii segments was increased up to 3-fold.The auxin uptake in stem-segments of the tumor-forming hybrid,however, could not be increased significantly by kinetin. Theeven distribution of IAA-¹⁴C in segments of normal and tumorproneNicotiana shoots is stimulated by kinetin. Data are discussedin conjunction with previous results on auxin transport andtumorformation in Nicotiana. (Received August 8, 1972; )     

Physiological activity of a viridicatin derivative, 3-(4-phenylcarbostyriloxy) acetic acid

A carboxymethylene derivative (V-OCH₂COOH) of viridicatin (V-OH)promoted the root growth of rice and sesame seedlings. V-OCH₂COOHhad no known hormonal activities, per se, but did have an inhibitoryeffect on IAA and 2,4-D-induced growth of Avena coleoptile sectionsand of carrot root callus. However, inhibition by VOCH2COOHof 2,4-D-induced growth in carrot root callus was to some extentreversed by increasing the concentration of 2,4-D. V-OCH₂C0OHseemed to competitively inhibit IAA-induced elongation of Avenacoleoptile sections. (Received September 14, 1970; )     

Somatic Embryogenesis and Analysis of Peroxidase in Cultured Lettuce (Lactuca sativa L.) Cotyledons

Somatic embryos were induced in lettuce cotyledons culturedon Murashige and Skoog's (MS) medium containing either 2 mgl^–1 6-benzylaminopurine (BA) and 0.2 mg l^–1 naphthaleneaceticacid (NAA) or 0.2 mg l^–1 BA and 2 mg l^–1 NAA. Bothcombinations induced a frequency of over 70%. The explants culturedonly in the presence of 2,4-dichlorphenoxyacetic acid (2,4-D)did not produce somatic embryos. The development of the embryoidswas studied histologically and by scanning electron microscopy.Peroxidase activity was assayed and the isoenzyme pattern ofcalluses was determined by polyacrylamide gel electrophoresis.Callus from an embryogenic line showed a much higher peroxidaseactivity than that from a non-embryogenic line, one extra peroxidaseisozyme band being present and typical of the embryogenic callus.No qualitative differences were detectable between the embryogeniccalluses. Lactuca sativa L, lettuce, somatic embryogenesis, peroxidases, isoenzymes     

Studies on the Growth in Culture of Plant Cells: XX. UTILIZATION OF 2,4-DICHLOROPHENOXYACETIC ACID BY STEADY-STATE CELL CULTURES OF ACER PSEUDOPLATANUS L

Omission of 2,4-dichlorophenoxyacetic acid (2,4-D) from batchcultures of sycamore produced an immediate reduction in ratesof cell division and eventually in rates of biomass accumulation.The sequential responses of a chemostat and of turbidostat culturessubjected to gradual withdrawal of 2,4-P were: (i) a transientincrease in biomass accumulation, (ii) increased accumulationof p-coumaric acid, flavonoids, and lignin, (iii) increasedcell aggregation, (iv) reduced rates of cell division, and (v)death. During stepwise reduction of 2,4-D supplied to turbidostatcultures, rates of 2,4-D uptake were reduced when the spentmedium concentration fell to 3?5–1?0 ? 10^–7 M. Underthese conditions the 2,4-D concentration in soluble and insolublecell fractions declined. The growth responses were correlatedwith the spent-medium 2,4-D concentration but not with its concentrationin the intracellular fractions.