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: 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 Uptake of Growth Substances: XVL. THE ENTRY OF CHLORINATED PHENOXY ACETIC ACIDS INTO THE EPIDERMAL AND CUT SURFACES OF ETIOLATED OR GREEN STEM SEGMENTS

A comparative study has been made of the patterns of entry,into segments with sealed and open ends from etiolated or greeninternodes of Pisumsativum, of phenoxyacetic acid (POA) andits 2-, 4-, 2, 6-, 2, 4, 5- and 2, 4, 6-chlorodenvativas, eachcontaining ¹⁴C in the carboxyl group. When the ends are sealed pretreatment in the light at a highintensity greatly suppresses the subsequent entry of the individualcompounds compared to pretreatment in the dark. During uptake,exposure to light or dark has no effect. There are also no significantdifferences in the level of accumulation between compounds. For segments with open ends pretreatment in the light has asmaller depressive influence. On the other hand, illuminationduring uptake brings about significant but not large increasesfor some but not all compounds. Under these conditions uptakeis determined by the nature of the compound. It is greatestfor POA and least for the 2, 4, 5- and 2, 4, 6-chloroderivatives. It is concluded that during pretreatment in the light increasingthe intensity induces a thicker cuticle. As a consequence forsegments with sealed ends the importance of the physical processesconcerned with passage through the cuticle are enhanced. Forsegments with open ends, when much of the entry is through thecut surfaces, active transport plays a more important role anda light response may be involved. It is, however, much smallerthan that recorded for the uptake of chloride ions.     

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: 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: 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: III. INFLUENCE OF INDOLEACETIC ACID AND OTHER AUXINS ON THE UPTAKE OF 2,4-DICHLOROPHENOXY-ACETIC ACID BY CHLORELLA

When 2,4-dichlorophenoxyacetic acid, labelled with ¹⁴C, is accumulatedby Chlorella pyrenoidosa, an appreciable fraction, which increaseswith time, is held within the cell in a form which is not removedwhen the cells are placed in a solution containing the unlabelledcompound. The accumulation of this fraction is dependent uponthe supply of metabolic energy since it is affected by temperature,light, and the addition of inhibitors or citrate to the externalmedium. The uptake of this metabolically accumulated 2,4-D follows apattern which indicates that a single enzymic reaction is apredominant component of the uptake process. In the presenceof the other auxins, including indoleacetic acid, 2,4,5-trichlorophenoxyaceticacid and 4-chlorophenoxyacetic acid, the uptake of 2,4-D isdepressed and the changes suggest that there is competitionat the site of this reaction. The nature of the competitionis seemingly of the type where each compound serves as a substratefor the enzyme. It may be significant that the extent to which the individualauxins compete with 2,4-D is in the same order as their generaleffectiveness as growth regulators for other species.     

Studies on Foliar Penetration: I. FACTORS CONTROLLING THE ENTRY OF 2, 4 - DICHLOROPHENOXYACETIC ACID

A technique, using leaf disks, has been developed to study thepenetration of isotopically labelled compounds into leaves underconditions where there is no appreciable change in the concentrationof the external solution and no subsequent translocation. Inthis preliminary survey, the leaves of Phaseolus vulgaris andColeus Blumei were employed to investigate the entry of 2,4-dichlorophenoxyaceticacid (2,4-D), labelled in the carboxyl group with ¹⁴C. Over3 days there is no loss of ¹⁴C to the atmosphere from treatedleaves of Phaseolus. The rate of penetration is enhanced when(a) the leaves are young, (b) the water status is lowered, (c)the temperature is raised (Q₁₀=2.3–2.8), and (d) a surface-activeagent is added to the external solution. Penetration is alsofavoured by a decrease in the pH, the relation indicating thatboth ions and molecules enter. Penetration is greater in thelight and prior illumination of the tissues positively affectsthe subsequent rate in the light, but not in the dark. In boththe light and the dark considerably more 2,4-D penetrates theabaxial surface of Phaseolus leaves. For Coleus an even greaterdifference between surfaces is found in the light but not inthe dark. For both species in the light the rates of entry intoboth surfaces are proportional to their respective stomataldensities. The simultaneous addition of indoleacetic acid tothe external solution caused more 2,4-D to enter Phaseolus leaves,but the addition of triiodobenzoic acid restricts entry. Therate of penetration remains constant over 24 hours and between0.1 and 200 mg./l. the rate is linearly related to concentration.Subsequent to entry, the 2,4-D is in a form which does not diffusefrom the tissue into buffer or exchange with unlabelled 2,4-D.Moreover, no outward movement takes place from treated tissuewhich has been frozen and thawed. These findings are discussedin relation to previous work on foliar penetration. It is concludedthat at least with Phaseolus penetration largely takes placethrough the guard cells and/or accessory cells.     

The Translocation of 3,5-Dichlorophenoxyacetic Acid in Relation to its Effect on Potato Common Scab

The translocation of 3,5-dichlorophenoxyacetic acid (3,5-D)in the potato plant was studied to clarify the mode of actionagainst potato common scab. 3,5-D was translocated to the tuberfaster and metabolised less than 2,4-D. Translocation to tuberswas proportional to growth and a concentration of 10–25nmol 3,5-D g^–1 fr. wt was attained 8 d after a foliarapplication. Previous evidence indicates this concentrationwas insufficient to be directly toxic to the pathogen Streptomycesscabies and the results suggest that 3,5-D acts in the tuberby altering the host's response to infection.     

The Effects of 2,4-Dichlorophenoxyacetic Acid on Ion Uptake by Maize Roots

The effects of the synthetic auxin and herbicide 2,4-dichlorophenoxyaceticacid (2,4-D) on K^$ and Cl^– uptake and H^$ release by youngexcised maize roots has been studied. Brief exposure to 2,4-D(0.01 mmol dm^–3) at pH 3.5 causes a large depolarizationof the electrical potential across the root plasma membranesand converts K^$ uptake to K^$ leakage into the bathing solution.These results can be explained by the increased H^$ permeabilityof the membranes induced by the weak acid 2,4-D. The depolarizationresults in a less favourable electrochemical potential gradientfor K^$ uptake across these membranes. These effects are notrelated to the auxin properties of 2,4-D as the nonauxin 3,5-dichlorophenoxyaceticacid (3,5-D) gives rise to similar effects. The relative depolarizationsinduced by a range of weak acids appear to be unrelated to theiroil/water partition coefficients. In contrast, on bathing the roots for longer periods in solutions(pH > 5) containing 2,4-D (0.01 mmol dm^–3) K^$ and Cl^–uptake and H^$ release are inhibited. These effects are not shownwith 3,5-D suggesting an auxin-linked action for 2,4-D. Alsothe electrical potential across the plasma membranes is onlyslightly depolarized so that a change in the electrochemicalpotential gradient cannot be invoked to explain the loweredion fluxes. The evidence is consistent with the removal of anenergy supply to a metabolically linked K^–/H^– exchangemechanism in the plasma membranes. It is likely that both modes of action would operate to lowerion uptake under soil-grown conditions, the former becomingmore manifest in acidic soils.     

Some Physiological Responses of Chlorella pyrenoidosa to 2,4-Dichlorophenoxyacetic Acid

An 18-h treatment of synchronously-grown Chlorella pyrenoidosawith 2,4-D did not significantly alter the size, dry weight,degree of synchrony, or pigment content of the cells, nor weredetectable quantities of ethylene produced. When Chlorella pyrenoidosawas treated with 5?10^–4 M 2,4-D, there was a statisticallysignificant stimulation of both net oxygen uptake and productionwhile 5?10 M 2,4-D inhibited both processes. When Chlorellapyrenoidosa was treated with 5?10^–4 M and 5?10^–3M 2,4-D, significantly greater amounts of glycollate were presentin the culture medium, even though an assay for glycollate dehydrogenaseshowed that the activity of this enzyme from 2,4-D-treated Chlorellapyrenoidosa was three times greater than in control cells. Looselybound 2,4-D was partitioned from a nonaqueously isolated chloroplastfraction, while other cell fractions failed to show detectablequantities of 2,4-D. It is postulated that in Chlorella pyrenoidosathe chloroplast is a target for 2,4-D action and that interferencein photorespiratory processes may underlie the observed responses.     

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.     

Studies on Foliar Penetration: VIII. EFFECTS OF CHLORINATION ON THE MOVEMENT OF PHENOXYACETIC AND BENZOIC ACIDS THROUGH CUTICLES ISOLATED FROM THE FRUITS OF LYCOPERSICON ESCULENTUM L.

The effects of chlorine substitution on the movement of phenoxyaceticand benzoic acids through enzymatically-isolated cuticles ofLycopersicon fruits were determined by following the transferof each acid containing ¹⁴C from a donor to a receiver solution.This cuticle is characterized by an isotropic cutin matrix,within which patches of birefringent cuticular waxes are foundnear the outer surface. The outer, morphological surface isrelatively smooth while at the junction with the outer wallsof the epidermal cells there is extensive cuticular developmentextending down between the anticlinal walls. The epicuticularwax appears as a soft sheet-like covering of which the surfaceis relatively featureless. Chlorination of phenoxyacetic acid results in an enhanced transferacross the isolated cuticle. The order was 2,4,5- and 2,4,6-trichlorophenoxyacetic> 2,4- and 3,5-dichlorophenoxyacetic > 2-chlorophenoxyacetic> phenoxyacetic acid. Removal of the epicuticular wax resultedin greater permeability for all compounds; transfer of the morepolar acids was favoured. In contrast, chlorination of benzoicacid decreases passage through the cuticle; the rate is highestfor benzoic acid followed in descending order by 2-chlorobenzoic,2,4- and 2,5-dichlorobenzoic and 2,3,6-trichlorobenzoic acid.Chlorination also depresses the passage of both phenoxyaceticand benzoic acid through a dialysis membrane. The effects ofchlorination on the lipid solubility of both series of compoundsare discussed in relation to differences in transfer acrossthe cuticle.     

Studies on Foliar Penetration: III. THE EFFECTS OF CHLORINATION ON THE RATE OF PENETRATION OF PHENOXYACETIC ACID AND BENZOIC ACID INTO LEAVES OF PHASEOLUS VULGARIS

A comparative study has been made of the penetration into leafdisks of Phaseolus vulgaris of (a) phenoxyacetic acid and its2-, 4-, 2, 4-, 2, 6-, 3-5, 2, 4, 5-, and 2, 4, 6- chloro derivatives,and (b) benzoic acid and its 2-, 2, 4-, 2, 5-, 2, 3, 6-chloroand 3, 6-dichloro-2-methoxy derivatives. The methods of synthesisof each compound with ¹⁴C incorporated in the carboxyl groupare described. In the series of substituted phenoxyacetic acids it was establishedthat only for 2, 4, 5-T was there an appreciable loss of radioactivityfrom the system either in the light or darkness. In contrast,with the exception of 2, 4-DCBA, ¹⁴C is lost from each memberof the series of substituted benzoic acids. The level and pattern of penetration in the two series is differentiallyaffected by chlorination. In general progressive chlorinationof phenoxyacetic acid leads to an increase in the rate of penetrationin both light and darkness. There are, however, exceptions;for illuminated disks the rate of entry of 2,4,5-T is exceededby 2,4,6-T, 2, 4-D, and 2, 6-D whereas in the dark 2,4,5-T hasby far the highest rate of penetration. Progressive chlorinationof benzoic acid, however, depresses entry in both light anddarkness. Possible relationships between these findings and changes inselected physical properties of each series of compounds havebeen examined. Between members of the series of substitutedphenoxyacetic acids there is little variation in the dissociationconstant whereas for the substituted benzoic acids there isa marked lowering of the pK as the degree of chlorination increases.The rate of elution with chloroform of each compound from abuffered Silocel column gives a measure of the degree of partitioninto a lipid medium at a given pH. At pH 5.2 chlorination ofphenoxyacetic acid results in more rapid elution whereas chlorinationof benzoic acid causes a longer hold up. An apparatus was designed to enable measurements to be madeof the rate at which a compound moves from one aqueous phaseto another through a layer of lipid. Chlorination promotes therate of transfer of phenoxyacetic acid through octanol but retardsthe transfer of benzoic acid. The extent to which chlorinationdepresses the rate of transfer of benzoic acid matches the diminutionin the rate of leaf penetration. For the series of substitutedphenoxyacetic acids transfer rates and penetration rates followsimilar trends in the dark but in the light agreement is lessgood. These findings are discussed in relation to prior studies onthe rate of uptake and the metabolic fate of the ndividual compoundsin a number of plant tissues.     

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.     

Wheat Callus Culture: the Initiation, Growth and Organogenesis of Callus Derived from Various Explant Sources

Callus was obtained from mature excised embryos of wheat, fromnodal and internodal stem segments and from rachis segmentsusing the medium of Murashige and Skoog(1962)(M medium), containing1-0mg l^–1 2,4-D, and from immature embryos using the mediumof Green and Phillips (1975) containing 2 mg l^–1 2,4-D.Callus yield from mature embryos depended upon the cultivarused. No callus could be obtained from leaf segments. Callusderived from mature embryos and nodal stem segments was successfullymaintained by serial sub-culture on the M medium containing2,4-D for up to 3 years although its growth rate declined toa lower level as culture proceeded. Such cultures consistently produced roots when transferred toa medium containing a low level of 2,4-D or no 2,4-D. The presenceof the auxin was essential for continued proliferation of thecallus tissue. Shoot initiation was infrequent, did not occurafter the first few sub-cultures and could not be enhanced byvarious auxin and cytokinin additions to the medium. Callusderived from immature embryos did not have an enhanced potentialfor shoot initiation. Triticum aestivum, wheat, callus culture, organogenesis     

Studies on Foliar Penetration: VI. FACTORS CONTROLLING THE PENETRATION OF 4-AMINO-3,5,6-TRI-CHLOROPICOLINIC ACID (PICLORAM) INTO THE LEAVES OF PHASEOLUS VULGARIS

The evaluation of the factors controlling the penetration of4-amino-3,5,6-trichloropicounic acid (picloram), containing¹⁴C in the carboxyl group, into leaves of Phaseolus vulgarisis complicated by the ability of the leaf tissue to decarboxylatethe compound rapidly. The degradation system becomes fully activatedonly after some hours following treatment with picloram whilethe capacity for decarboxylation declines with the age of theleaf. When allowance for decarboxylation is made, the pattern of penetrationfor picloram resembles in particular that of 2,2-dichloropropionicacid (dalapon). At the abaxial surface penetration continuesover 24 h at a fairly constant rate but in light, which enhancespenetration, a ‘surge’occurs between the secondand sixth hour. Raising the external concentration promotespenetration and in light entry is directly proportional to concentrationup to at least 2.5 x 10^–l M. The response to varying lightintensity resembles that of dalapon rather than 2,4-D. Evenlow intensities promote penetration; the effect is maximal atabout 10 000 lx at the abaxial surface but continues at leastup to 20 000 lx at the adaxial surface. Penetration is retarded at 4°C but the decarboxylation systemremains functional. In light, but not in darkness, penetrationis strikingly reduced by 3-(3,4-dichlorophenyl)-l,l-dimethylurea(DCMU). Raising the external pH from 4.2 to 7.2 retards entry. It is concluded that the factors which determine the penetrationof picloram into Phaseolus leaves also control the entry ofa wide range of organic compounds and inorganic ions.     

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.     

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.     

Somatic Embryogenesis and Plant Regeneration from Cultured Leaf Explants of Zea mays

Young leaf segments of Zea mays L. seedlings were cultured onMurashige and Skoog's basal nutrient medium supplemented with2 mg l^–1 2, 4-D and sub-cultured on medium containing8 mg l^–1 2,4-D. Two types of callus tissues appeared—embryogenicand non-embryogenic. The embryogenic callus tissue producednumerous somatic embryos which on transfer to media containinglow amounts of 2,4-D or ABA produced plantlets. Callus tissuesexhibited embryogenic potential for more than 1 year. Zea mays L. cv. Ageti-76, Zea mays L. cv. N-L-D-Comp., maize, leaf, callus, somatic embryogenesis, regeneration