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: 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: 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: 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.     

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.     

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.     

Studies on Foliar Penetration: VII. FACTORS CONTROLLING THE PENETRATION OF CHLORIDE IONS INTO THE LEAVES OF PHASEOLUS VULGARIS

The pattern of penetration of chloride ions into the abaxialsurfaces of the primary leaves of Phaseolus vulgaris has severalfeatures in common with those previously recorded for 2,4-dichlorophenoxyaceticacid (2,4-D), 2,2-dichloropropionic acid (dalapon), and 4-amino-3,5,6-trichloropicolinicacid (picloram). In the dark the rate of entry of chloride ionsup to 24 h is constant, but in the light entry is at first slowand then more rapid. This acceleration does not occur at lowtemperature or when the tissue is treated with 3-(3,4-dichlorophenyl)-l,l-dimethylureaor phenylmercuric chloride. Neither does it take place at theadaxial surface or when the leaves are more mature. The most distinguishing feature of the pattern of penetrationof chloride ions is its dependence on external pH. In darknessentry is unaffected by changes in pH but in the light the rateof entry is increased as the pH falls, but this response isrestricted to young leaves. No binding of chloride seems tooccur within the tissue. These findings support the view that penetration of the abaxialsurface in young leaves of Phaseolus is largely determined bya membrane system. This system decreases in importance as theleaf matures and the overlying cuticle thickens. At the adaxialsurface the thicker cuticle is seemingly a major barrier topenetration even in very young leaves.     

The Metabolic Fate of Phenoxyacetic Acids in Higher Plants

The metabolism of phenoxyacetie acid (POA) was followed overshort periods in segments of Auena and Pisum using a vacuuminfiltration technique. Both species form 4-hydroxyphenoxy-aceticacid (4-OH-POA) and its phenolic glucoside (OGlu-POA) but Pisumincorporates much of the substrate into an insoluble wall component.This insoluble material appears to be formed from 4-OH-POA.Preliminary kinetic experiments show that the overall conversionof POA is linearly related to concentration up to 2µmolPOA g^-1 fresh tissue and can be completed in a few hours. Themethod provides a very useful means of preparing sufficientquantities of ¹⁴C-labelled 4 OH POA and O-Glu-POA in a pureform for subsequent use as substrates for the enzymes involvedin glucosylation and formation of the insoluble material. Althoughlight has relatively little effect on hydroxylation, the capacityformation of both the glucoside and the insoluble material isincreased by continuous fluorescent, red, and far red light.The results so far obtained suggest that phytochromeis involvedin the transformations. The chlorinated derivatives of phenoxyaceticacid with high herbicidal activity e.g. 2, 4-dichlorophenoxyaceticacid (2, 4-D) are also metabolized by analogous reactions andthe formation of hydroxylated and glycosylated derivatives maycontribute to the detoxification of such herbicides in resistantspecies.     

EFFECTS OF CHLORAMPHENICOL AND KINETIN ON UPTAKE AND INCORPORATION OF AMINO ACIDS BY TOBACCO LEAF DISKS

The effects of chloramphenicol and kinetin on uptake and incorporationof ³⁵S-methionine and some ₁₄C-amino acids have been investigatedin leaf-disks of Nicotiana rustica in light and dark. Chloramphenicolin a concentration of 1 mg per ml inhibits the uptake of aminoacids from 30 to 60 per cent compared with the water control.The incorporation of amino acids into bulk protein is stronglyinhibited in light (40 to 70 per cent), but only to a smalldegree in dark (10 to 20 per cent), as revealed also by ¹⁴CO₂-photosynthesisof the disks and following treatment with chloramphenicol indark. The stimulating effect of kinetin on uptake and incorporationof amino acids is dependent upon its concentration (10^–5to 10^–6 M ; but 10^–4 M solution inhibits stronglyboth uptake and incorporation). The stimulation seems to influencemore incorporation than uptake processes. Possible interactionsof chloramphenicol and kinetin in the protein metabolism oftobacco leaves have been discussed. (Received April 27, 1964; )     

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 Diurnal Pattern of Nitrate Uptake and Reduction by Spinach (Spinacia oleracea L.)

Spinach plants were grown in bowls of aerated nutrient solutionin a controlled environment chamber for 24 h, and harvestedevery 3·5-5 h to record their growth, nitrate and wateruptake, and plant nitrate concentration. Twelve such experimentsare described, either with a 14/10 h dark/light regime, or continuouslight or darkness. The irradiance was either 110, 320, or 510µmol m^-2 s^-1 (PPFD). All these regimes began at the endof the light period of a 14/10 h dark/light regime (510 µmolm^-2 s^-1) lasting approximately 2 weeks. Nitrate uptake rate per g of dry weight of plant continued almostunabated at about 17 µmol h^-1 through the initial 14-hdark period, and then fell away sharply if the light was notrestored, but increased slightly when it was. With continuouslight at 510 µmol m^-2 s^-1, uptake rate rose steadily forthe first 24 h of light, and then fell sharply for about 6 h.Shoot nitrate concentration increased about three-fold in thedark phase, and declined in the light at a rate which was positivelyrelated to the irradiance. Root nitrate concentration was severaltimes higher than that of the shoot: its diurnal change wassmaller (relative to the mean) than that of the shoot. Nitratereduction occurred to a small extent in the dark, and increasedrapidly as soon as the lights came on, to remain at a roughlyconstant rate (related to the irradiance) throughout the lightphase. Dry matter increase in the light was related to irradiance,but with little increase above 320 µmol m^-2 s^-1. Respiratoryweight loss in the dark was not detectable. Rate of fresh weightincrease was approximately constant throughout light and darkperiods. The results compare quite well with the predictions of a simplesimulation model, based on the pump/leak principle.Copyright1994, 1999 Academic Press Spinacia oleracea, nitrate, uptake, reduction, influx, efflux, diurnal, regulation, model, simulation     

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.     

Induction of Light Dependent Pyruvate Transport into Chloroplasts of Mesembryanthemum crystallinum by Salt Stress

Mode of photosynthesis in Mesembryanthemum crystallinum changesfrom C₃ to Crassulacean acid metabolism (CAM) when the plantswere stressed with high salinity. [¹⁴C]Pyruvate uptake for 30s into intact chloroplasts isolated from leaves of the CAM modeof M. crystallinum was enhanced more than 5-fold in the lightcompared with that in the dark. The stromal concentration ofpyruvate in the light reached to more than 2.5 times of themedium. In contrast, little or no pyruvate uptake occurred inchloroplasts from C₃ leaves in either light or dark condition.The initial uptake rate (10 s incubation at 4°C) into theCAM chloroplasts in the light was about 3-fold higher than therate in the dark. K_m and V_max of the initial uptake in the lightwere 0.54 mM and 8.5 µmol (mg Chl)^–1 h^–1 respectively.These suggest that pyruvate was actively incorporated into theCAM chloroplasts against its concentration gradient across theenvelope in the light. When hydroponically grown M. crystallinumwere stressed by 350 mM NaCl, the capacity of chloroplasts forpyruvate uptake was induced in 6 d corresponding to the inductionof the activities of PEP-carboxylase and NAD(P)⁺-malic enzymesin response to salt stress. (Received October 12, 1995; Accepted January 19, 1996)     

STUDIES ON THE RE-ASSIMILATION OF RESPIRATORY CO2 IN ILLUMINATED LEAVES

Experiments were performed, using rice, barley and Hydrangealeaves, to examine the re-assimilation of respiratory ¹⁴CO₂while photosynthesis is going on in an open air flow system. It was found that the leaves which had assimilated ¹⁴CO₂ beforehandevolved, when kept under photosynthesizing conditions, threeto four tenths (variable according to plant species and externalconditions) of the amount of ¹⁴CO₂ to be produced in the dark.Such an incomplete re-utilization of ¹⁴CO₂ was observed alsoin spinach leaf homogenate as well as in the leaves which hadpreviously absorbed ¹⁴C-glucose. The ¹⁴CO₂ output in rice leaves was found to be acceleratedby the light of high intensity. A possibility of light stimulationon the respiration was suggested. (Received October 7, 1961; )     

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: I. FACTORS CONTROLLING THE UPTAKE OF PHENOXYACETIC ACIEDS BY LEMNA MINOR

An analysis has been made of the factors controlling the uptakeof 2:4-dichloro-phenoxyacetic acid (2:4-D) and related compoundsby Lemna minor, grown under controlled conditions. All compoundswere labelled with ¹⁴C in the carboxyl group and the ¹⁴C inthe plants, after liquid combustion, assayed as barium carbonate. With 2:4-D the rate of entry from concentrations of 8 to 32mg./l. is maximal in the first 20 minutes, then falls progressivelyuntil the rate is zero between 1 and 2 hours and in the thirdphase (up to 24 hours) there is a net loss from the plants dueto an outward movement of the compound back into the externalsolution. When the chlorine substitutions are in the 2:6-positionthe course of uptake is similar, save that loss to the solutiontakes place later. For the 2-chloro-substitution, after theinitial phase of uptake there is some loss but this is followedby a period of recovery and uptake again proceeds, but slowly.In contrast, the course of uptake over 24 hours of phenoxyaceticacid is normal, i.e. there is a steady accumulation. When plants are pretreated with labelled 2:4-D for 30–60minutes and transferred to water or culture solution then over90 per cent. of the ¹⁴C is found in the external medium after4:5 hours and this release cannot be accounted for in termsof exchange processes since the addition of unlabelled compoundto the medium retards the rate of loss. This loss is slowedbut not stopped at 1.25°C. and up to 22.5°C. the rangeof the Q₁₀ is 1.6–1.9. Uptake in the first 30 minutes is temperature sensitive between7.5 and 30°C. (Q₁₀ 2.3–2.6) and in general is positivelyand curvilinearly related to the external concentration. Pretreatmentwith unlabelled compound up to 2 hours progressively depressesthe subsequent initial uptake of labeled growth regulator. Itis concluded that initially the rate of entry greatly exceedsthe rate of loss but that with time the ratio steadily diminishesto less than unity. Initial uptake of 2:4-D is markedly dependent on the pH of thesolution and closely but not completely correlated with theexternal concentration of undissociated molecules. On the otherhand, the outward movement is relatively unaffected by the externalpH. Combinations of concentration and time of exposure which bringabout loss to the solution need not cause any permanent retardationof growth. In fact, exposure for 1 hour to 8 mg./l. significantlyaccelerated growth in the next 8 days. These results are discussed in relation to previous findingsand it is concluded that the pattern of uptake is determinedon the one hand by the chemical structure of the growth substanceand on the other by specific physiological differences at celllevel.     

Links between Glucose Uptake and Metabolism in Nitella translucens

The uptake of ¹⁴C-glucose into cells of Nitella translucenshas been investigated under experimental conditions previouslyused in studies of the ionic relations of these cells. Glucoseentry was considerably stimulated by light, and under aerobicconditions the fluxes remained constant for many hours. Theinflux of glucose was inhibited by over 80 per cent at low temperature(4° C) and by over 90 per cent by the uncoupler carbonylcycanide-m-chlorophenylhydrazone. 2-Deoxy-D-glucose was a non-competitiveinhibitor of glucose uptake both in light and darkness. Cyclicphotophosphorylation promoted the influx (with decreasing efficiency)for several hours. It is suggested that an ATP-dependent transportprocess controls glucose entry to the cells, and that passivediffusion is of little significance.     

The Photometabolism of Acetate by Chlorella pyrenoidosa

Chlorella pyrenoidosa can utilize sodium acetate as a carbonsource for growth in the light. Growth proceeds under aerobicconditions both in the presence and in the absence of carbondioxide, but under anaerobic conditions only in its presence.The assimilation of acetate does not result from oxidation tocarbon dioxide followed by photosynthetic fixation because theproducts of ¹⁴C-acetate assimilation are different from theproducts of ¹⁴CO₂ fixation in the presence of unlabelled acetate. In aerobic conditions 10-⁶ M DCMU induces a pattern of acetateassimilation in the light similar to that in the dark. Thus,in the presence of DCMU in the light, less acetate carbon isincorporated into cells, particularly into lipids, polysaccharide,and protein, and more is released as carbon dioxide than inits absence. The effect of 4 x 10^-3 M MFA on acetate assimilationin the presence of 10^-6 M DCMU is the same in light and dark.Acetate assimilation is unaffected by desaspidine and sodiumbisulphite. The mean generation time of C. pyrenoidosa growing on acetatein the light under aerobic conditions is 20 hours. When 10^-5M DCMU is added the mean generation time is 60 hours, the sameas that for Chlorella growing on acetate in the dark. The activityof the enzymes of the glyoxylate cycle, isocitrate lyase (E.C.4.1.3.1.)and malate synthetase (E.C.4.1.3.2.) is repressed in the light,but activity of both enzymes increases markedly when DCMU isadded.     

Dark uptake of inorganic14C in oligotrophic oceanic waters

Dark uptake of inorganic ¹⁴C by offshore plankton was measuredat two depths at 36 stations in the Atlantic Ocean from 52°Sto 26°N, mainly along 30°W. The samples were incubatedfor 2 h with and without inhibition of biological activity withHgCl₂. In addition, six time course experiments were performed.The mean dark uptake rate varied from 0.68 to 4.82 (µmolC m^–3 h^–1 over the transect and showed a significantpositive relationship with chlorophyll a. The dark uptake wasusually >5% of the maximum photosynthetic capacity (P^m),and higher values relative to P^m were associated with low valuesof P_m and not with high absolute dark values. A linear relationshipbetween dark uptake and P_m was found with a background value(y-axis intercept) of 0.51 (µmol C m^–3 h^–1and a slope of 0.77% of P^m. A major fraction of the dark signal,66–80% of the total signal, persisted in bottles treatedwith HgCl2, indicating that most of the dark signal was independentof biological activity. Time course experiments showed a lineardark uptake with time for the first hours, whereafter the uptakeceased. At stations with low concentrations of inorganic nitrogen[>1 (µmol (NH₄⁺+NO₃^–)], a second stage was observedafter 3–8 h, probably due to an increase in bacterialactivity. The results suggest three mechanisms for the darkvalue in short-term incubations in oligotrophic waters. A backgroundvalue independent of biomass and incubation time which was thedominant part of the dark signal in samples with very low phytoplanktonbiomass (>0.3 p-g Chi a 1"). Another important part was residualsof ¹⁴C associated with plankton, probably adsorbed to compoundsinside the cells. This fraction was dominant in short-term incubationsat chlorophyll concentrations >0.3 p.g Chi a H. Active uptakeby living cells (total minus ‘HgCl2 uptake‘) wasonly a minor part of the dark signal in short-term incubations,but dominated at longer incubation time (>3–9 h), probablydriven by an increase in bacterial activity. A significant enhancementof the non-photosynthetic uptake of ¹⁴C was observed in light,probably associated with a carbon-concentrating mechanism inphytoplankton or light stimulation of ß-carboxylationactivity. The results strongly suggest that dark values shouldbe subtracted from the light uptake. This correction is particularlyimportant when photosynthetic rates are low, e.g. at low lightor in short-term incubations where a time-zero background becomesa significant part of the total uptake in light. Present address: National Environmental Research Institute,Department of Marine Ecology and Microbiology, Frederiksborgvej399, PO Box 358, DK-4000 Roskilde, Denmark