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.     

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.     

Measurement of intracellular and extracellular free calcium in apple fruit cells using calcium-selective microelectrodes

Abstract. Intracellular and extracellular free calcium was measured in cortical cells from apple fruit ( Malus domestica Borkh cv. Cox's Orange Pippin) using calcium-selective microelectrodes. It was difficult to position microelectrodes in the cytoplasm, and thus most intracellular measurements reflected vacuolar concentrations of about 0.6mol m⁻³ free calcium. Extracellular free calcium was measured in wells (0.2mm³) that had been cut through the epidermis of the fruit, then filled with osmoticum. Concentrations of free calcium in the sampling well varied between 0.02 and 1.3 mol m⁻³, and were related to the calcium content of the tissue. Functioning of the plasma membrane, and perhaps the supply of calcium for intracellular needs during fruit ripening and storage, may require mol m⁻³ concentrations of extracellular free calcium. Thus, it is suggested that low levels of extracellular free calcium in calcium-deficient fruit may be partly responsible for development of deficiency disorders.     

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.