Channel-mediated K+ flux in barley aleurone protoplasts

Gibberellic acid (GA₃) stimulates K⁺ efflux from the barley (Hordeum vulgare L. cv. Himalaya) aleurone. We investigated the mechanism of K⁺ flux across the plasma membrane of aleurone protoplasts using patch-clamp techniques. Potassium-ion currents, measured over the entire surface of the protoplast plasma membrane, were induced when the electrochemical gradient for K⁺ was inward (into the cytoplasm). The magnitude and voltage-dependence of this inward current were the same in protoplasts treated with GA₃ and in control protoplasts (no GA₃). Inward currents activated by negative shifts in the membrane potential (E_M) from the Nernst potential for K⁺ (E_K) showed membrane conductance to be a function of the electrochemical gradient (i.e. E_M-E_K). Single-channel influx currents of K⁺ were recorded in small patches of the plasma membrane. These channels had a single-channel conductance of 5–10 pS with 100 mM K⁺ on the inside and 10 mM K⁺ on the outside of the plasma membrane. Single-channel currents, like whole-cell currents, were the same in protoplasts treated with GA₃ and control protoplasts. Voltage-gated efflux currents were found only in protoplasts tha thad been incubated without GA₃. We conclude that K⁺ influx in the aleurone is mediated by channels and these membrane proteins are not greatly effected by GA₃.Abbreviations and symbols F_K Nernst potential for K⁺ - E_M membrane potential - E_rev reversal potential - GA₃ gibberellic acid - K_i concentration of K⁺ inside the cell - K_o concentration of K⁺ outside the cell - R gas constant - S conductance (siemens) - T temperature (^oK) - _i ionic activity coefficient for internal (cytoplasmic) solution - _o ionic activity coefficient for external medium     

Metabolism of the Fungicide S-n-Butyl S′-p-tert-Butylbenzyl N-3-Pyridyldithiocarbonimidate (S-1358) in Rats

S-1358 was rapidly absorbed, metabolized and readily excreted via urine and feces from orally dosed rats. Excretion of radioactivity was almost complete within 4 days. The radioactivity was distributed mainly in stomach, intestines, liver and kidneys. It seems that S-1358 and its metabolites do not persist in organs and tissues following a single oral dosing.

Major urinary metabolites of the benzyl-labeled S-1358 were p-(1,1-dimethyl-2-hydroxyethyl)benzyl methyl sulfide [B], p-(1,1-dimethyl-2-hydroxyethyl)benzyl methyl sulfone [A], p-(1-methyl-1-carboxylethyl)benzyl methyl sulfide [D], p-(1-methyl-1-carboxylethyl)benzyl methyl sulfone [C] and their glucuronide conjugates. Fecal metabolites were S-n-butyl S′-(1, 1-dimethyl-2-hydroxyethyl)benzyl N-3-pyridyldithiocarbonimidate [MR], A, B, C and D. These metabolites were also found in the bile. The pyridine-labeled S-1358 gave rise to 2-(3′-pyridylimino)-4-carboxylthiazolidine [HM] and 3-aminopyridine [AP] in the urine, and MR and AP in the feces. Intact S-1358 was a major component of the fecal radioactivity.     

Characterization of monoclonal antibodies to protoplast membranes of Nicotiana tabacum identified by an enzyme-linked immunosorbent assay

Murine monoclonal antibodies to protoplast membrne antigens were generated using mouse myelomas and spleen cells from mice immunized with Nicotiana tabacum L. leaf protoplasts. For selecting antibody-secreting clones, a sensitive and rapid enzyme-linked immunosorbent assay (ELISA) for monoclonal antibody binding to immobilized cellular membrane preparations or immobilized protoplasts was developed. With intact protoplasts as immobilized antigen, the ELISA is selective for antibodies that bind to plasma-membrane epitopes present on the external surface of protoplasts. Using the membrane ELISA, a total of 24 hybridoma lines were identified that secreted antibodies to plant membrane epitopes. The protoplast ELISA and subsequent immunofluorescence studies identified four hybridoma lines as secreting antibodies which bound to the external surface of protoplasts and cells. The corresponding antigens were not species- or tissue-specific, were periodatesensitive, and were located in membranes which equilibrated broadly throughout a linear sucrose gradient. When protein blots of electrophoretically separated membrane proteins were probed with these antibodies, a band of M_r 14 kilodaltons (kDa) and a smear of bands of M_r 45–120 kDa were labeled. An additional set of three antibodies appeared by immunofluorescence to bind to the plasma membrane of broken but not intact protoplasts and labeled membranes equilibrating at a density of approx. 1.12 kg·l^-1 in a linear sucrose density gradient. These classes of monoclonal antibodies enlarge the library of monoclonal antibodies (Norman et al. 1986, Planta 167, 452–459) available for the study of plant plasma-membrane structure and function.Abbreviations ELISA Enzyme-linked immunosorbent assay - Ig immunoglobulin - kDa kilodalton - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Monoclonal antibodies directed against protoplasts of soybean cells

Splenocytes, derived from mice that had been immunized with protoplasts prepared from suspension cultures of root cells of Glycine max (L.) Merr. (SB-1 cell line), were fused with a murine myeloma cell line. The resulting hybridoma cultures were screened for the production of antibodies directed against the soybean protoplasts and were then cloned. One monoclonal antibody, designated MVS-1, was found to bind to the outer surface of the plasma membrane on the basis of several criteria: (a) agglutination of the protoplasts; (b) binding of fluorescence-labeled immunoglobulin on protoplasts yielding a ring staining pattern with prominent intensity at the edges; and (c) saturable binding by protoplasts of ¹²⁵I-labeled Antibody MVS-1. The antigenic target of Antibody MVS-1, identified by immunoblotting techniques, contained a polypeptide of relative molecular mass (M_r) approx. 400000 under both reducing and non-reducing conditions. When the antigenic target of Antibody MVS-1 was chromatographed in potassium phosphate buffer, the position of elution corresponded to that of a high-molecular-weight species (M_r 400000). These results provide the protein characterization required for the analysis of the mobility of Antibody MVS-1 bound to the plasma membrane of SB-1 cells.Abbreviations D diffusion coefficient - M_r relative molecular mass - PBS phosphate-buffered saline (8.00 g NaCl, 1.15 g Na₂HPO₄, 0.20 g NaH₂PO₄ per 1 L, pH 7.2) - TPBS phosphate-buffered saline containing 0.5% Tween-20 - TX-100, TX-114 Triton X-100, X-114 - SDS sodium dodecyl sulfate     

Effects of plant growth regulators and other compounds on flow of latex in Hevea brasiliensis

Eighty-nine compounds have been tested for their ability to promote the flow of latex from Hevea brasiliensis after tapping. The twenty-four substances which showed significant activity were: twelve chloro- and fluoro-phenoxy alkylcarboxylic acids substituted in the 4-, 2, 4- and 2, 4, 5- positions, two chlorinated benzoic acids, α- and β-naphthaleneacetic acid, 3-indolylacetic acid, 2-methoxy-4-diethylamino-6-isopropylamino-1, 3, 5- triazine (methoxy-ipazine), N,N-diallyl-α-chloroacetamide (CDAA), 1,1′-dimethyl-4,4′-bi-pyridilium-2A (paraquat), neomycin, dichloro-m-xyienol, and phenyl mercury acetate and chloride. Despite the many replications the high variability of the yield data does not allow of a precise classification of the relative activities. The most active compounds were 2, 4-dichloro-5-fluorophenoxyacetic acid and 2, 4, 5-trichlorophenoxyacetic acid (2, 4, 5-T), although 2, 4-dichlorophen-oxyacetic acid (2, 4-D) gave increases in yield comparable to those of 2, 4, 5-T in some of the trials.     

Evidence for two different herbicide-binding proteins at the reducing side of Photosystem II

The binding behaviour at the thylakoid membrane of the radioactively labelled phenolic inhibitors 2-iodo-4-nitro-6-[2′,3′-³H]isobutylphenol and 3,5-diiodo-4-hydroxy[U-¹⁴C]benzonitrile (ioxynil) has been studied. As judged from displacement experiments with other herbicides, phenolic herbicides and herbicides as represented best by 3-(3,4-dichlorophenyl)-1,1-dimethylurea have different binding sites at the reducing side of Photosystem II. The binding parameters of phenolic herbicides are not, or only slightly, affected by trypsin treatment of chloroplasts. In chloroplasts, besides free pigments, lipids, and the light-harvesting chlorophyll $\text{a}\text{b}$ protein complex, a protein of molecular weight 41 000 is radioactively labelled by the photoaffinity label 4-nitro-2-azido-6-[2′,3′-³H]isobutylphenol. The amount of radioactivity bound to the 41 kDa protein is diminished if chloroplasts are incubated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea prior to addition of the photoaffinity label, but not if the 2,4-dinitrophenyl ether of 2-iodo-4-nitrothymol is used instead. These two compounds are characteristic representatives of inhibitiors acting at the reducing or the oxidizing site of plastoquinone, respectively. Based on these results, a model for two different herbicide-binding proteins at the reducing side of Photosystem II is presented.     

Callus proliferation from leaf protoplasts using phenylurea type cytokinin, 4-PU,inBetula platyphylla VAR.Japonica

Summary Protoplasts were isolated from leaves ofBetula platyphylla var.japonica using a 0.6M mannitol solution containing 1% Cellulase Onozuka R-10 and 1% Driselase. The cell division and colony formation were largely enhanced using Murashige and Skoog (1962) liquid medium at half strength (1/2 MS), containing 0.6M mannitol, 0.09M sucrose, and factorial combinations of 0.1–30 μM N-(2-chloro-4-pyridyl)-N′-phenylurea (4-pu) and 0.1–10 μM 1-naphthaleneacetic acid (NAA) or 0.1–30 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The optimal protoplast density was 5–7 × 10⁴/ml. Continuous callus proliferation from protoplasts was achieved by transferring colonies to fresh 1/2 MS agar medium containing 1 μM NAA and 1 μM 4-pu with no mannitol. It appeared that supplementation of the medium with phenylurea type cytokinin, 4-pu gave the successful callus proliferation from the protoplasts ofB. platyphylla.     

Synthesis of the 2′-,3′-Didehydro-2′-,3′-dideoxy and 2′-,3′-Dideoxy Derivatives of 6-Azauridine and a New Route to 2′-,3′-Didehydro-2′-,3′-dideoxy-5-chlorouridine

Abstract

The 5′-O-(4,4′-dimethoxytrityl) and 5′-O-(tert-butyldimethylsilyl) derivatives of 2′-,3′-O-thiocarbonyl-6-azauridine and 2′,3′-O-thiocarbonyl-5-chlorouridine were synthesized from the parent nucleosides by reaction with 4, 4′-dimethoxytrityl chloride and tert-butyldimethylsilyl chloride, respectively, followed by treatment with 1,1′-thiocarbonyldiimidazole. Introduction of a 2′-,3′-double bond into the sugar ring by reaction of the 5′-protected 2′-,3′-O-thionocarbonates with 1, 3-dimethyl-2-phenyl-1, 3, 2-diazaphospholidiine was unsuccessful, but could be accomplished satisfactorily with trimethyl phosphite. Reactions were generally more successful with the 5′-silylated than with the 5′-tritylated nucleosides. Formation of 2′-,3′-O-thiocarbonyl derivatives proceeded in higher yield with 5′-protected 6-azauridines than with the corresponding 5-chlorouridines because of the propensity of the latter to form 2,2′-anhydro derivatives. In the reaction of 5′-O-(tert-butyldimethylsilyl)-2′-,3′-O-thiocarbonyl-6-azauridine with trimethyl phosphite, introduction of the double bond was accompanied by N³-methylation. However this side reaction was not a problem with 5′-O-(tert-butyldimethylsilyl)-2′-, 3′-O-thioarbonyl-5-chlorouridine. Treatment of 5′-O-(tert-butyldimethylsilyl)-2′-, 3′-didehydro-2′-,3′-dideoxy-6-azauridine with tetrabutylammonium fluoride followed by hydrogenation afforded 2′-,3′-dideoxy-6-azauridine. Deprotection of 5′-O-(tert-butyldimethylsilyl)-2′-, 3′-didehydro-2′-,3′-dideoxy-5-chlorouridine yielded 2′-,3′-didehydro-2′-,3′-dide-oxy-5-chlorouridine.     

Photosynthesis involvement in the mechanism of action of diphenyl ether herbicides

Photosynthesis is not required for the toxicity of diphenyl ether herbicides, nor are chloroplast thylakoids the primary site of diphenyl ether herbicide activity. Isolated spinach (Spinacia oleracea L.) chloroplast fragments produced malonyl dialdehyde, indicating lipid peroxidation, when paraquat (1,1′-dimethyl-4,4′-bipyridinium ion) or diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea] were added to the medium, but no malonyl dialdehyde was produced when chloroplast fragments were treated with the methyl ester of acifluorfen (methyl 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoic acid), oxyfluorfen [2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-(trifluoromethyl)benzene], or MC15608 (methyl 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-chlorobenzoate). In most cases the toxicity of acifluorfen-methyl, oxyfluorfen, or MC15608 to the unicellular green alga Chlamydomonas eugametos (Moewus) did not decrease after simultaneous treatment with diuron. However, diuron significantly reduced cell death after paraquat treatment at all but the highest paraquat concentration tested (0.1 millimolar). These data indicate electron transport of photosynthesis is not serving the same function for diphenyl ether herbicides as for paraquat. Additional evidence for differential action of paraquat was obtained from the superoxide scavenger copper penicillamine (copper complex of 2-amino-3-mercapto-3-methylbutanoic acid). Copper penicillamine eliminated paraquat toxicity in cucumber (Cucumis sativus L.) cotyledons but did not reduce diphenyl ether herbicide toxicity.     

The effect of paraquat on flax cotyledon leaves: Physiological and biochemical changes

Summary Some of the physiological and biochemical changes which were found to occur during paraquat (1,1-dimethyl-4,4-bipyridylium ion) treatment of cotyledon leaves of Linium usitatissimum, are reported. Results showed an inhibition of photosynthetic CO₂ uptake and electron flow of isolated chloroplasts. An increase in membrane permeability, changes in the level of the major lipid components and of malondialdehyde. These are correlated with ultrastructural changes and the discussion includes a proposed mode of action for the herbicides.     

Adaptive Use of N2-Dimethyl-Substituted Pterins by Cultures of Crithidia fasciculata1

The compound 6-(L-erythro-1,2′,3′-trihydroxypropyl)pterin, at a concentration of 50 pg/ml (“L-erythro-neopteria”), supports half-maximal growth of Crithidia fasciculata; biopterin at a concentration of 30 pg/ml is shown to yield similar growth. N²-dimethyl-6-(L-erythro-1′,2′,3′-trihydroxypropyl)pterin (A) was inactive even at 100 ng/ml. Synergism was observed with the N²-dimethylamino derivative (A) in the presence of suboptimal biopterin, its activity then being of the order of L-erythro-neopterin. In contrast, the stereoisomeric N²-dimethyl-6-(D-erythro-1′,2′,3′-trihydroxypropyl)pterin (“dimethyl-D-erythro-neopterin”) and its 3′-mono-phosphate only slightly enhanced growth under similar conditions but both threo-isomers had no supplementary activity. Biopterin-induced growth was slowed by 6-(D-erythro1′,2′,3′-trihydroxypropyl)pterin (D-neopterin); the threo-isomers had no such effect. An adaptive demethylation capacity by growing cultures and competition of biopterin uptake by D-neopterin seems likely. The report of the occurrence in Euglena of N₂-dimethyl-6-(L-threo-1′,2′,3′-trihydroxypropyl)pterin and its 3′-mono-phosphate adds further interest to our observations.     

Brain Pharmacokinetics of Non‐Imidazole Biphenyl H3 Receptor Antagonists: a Liquid Chromatography/Electrospray‐Mass Spectrometry and ex vivo Binding Study in Rats

In the present article, we report on the kinetics of brain penetration in rats of the H3R antagonist 1,1′‐[1,1′‐biphenyl‐4,4′‐diylbis(methylene)]bis‐[piperidine] ( 1 ), which had shown a favorable in vitro pharmacological profile and in vivo potency in preventing scopolamine‐induced amnesia. Two different approaches were employed: high‐performance liquid chromatography/electrospray‐mass spectrometry (HPLC/ESI‐MS) and ex vivo binding against the labeled agonist [³H]‐(R)‐α‐methylhistamine ([³H]RAMHA). Starting from the structure of 1 , the rigid piperidine ring was replaced by a flexible dipropylamino group (see 2 ) or by a morpholino ring (see 3 ), endowed with lower basicity. The effect of replacement on rat plasma and brain disposition in the 24 h after administration was analyzed. High (μM ) and persistent concentrations of 1 were found in rat plasma, while plasma levels were significantly lower (range: 0–200 nM ) for the other two derivatives. This could be explained, among other factors, by the higher stability, observed for 1 , to liver metabolic cleavage. The applied chemical modulation had an important effect on in vivo brain disposition, as, despite the comparable physico‐chemical properties, 2 did not show the tendency to accumulate within the brain, as stated by its brain vs. plasma concentration ratios, if compared to 1 . These structure? property relationships should be taken into account in the pharmacokinetic optimization of new series of H3 receptor antagonists.     

Effect of DCMU, Simazine and Atrazine on Nitrate Reductase Activity in Hordeum vulgare in vitro

The effect of three herbicides—DCMU (1,1-dimethyl-3- (3,4-dichlorophenyl) -urea), Simazine (2,4-bis(ethylamino)- 6-chloro-s-triazine), and Atrazine (2-chloro-4-ethylamino-6-iso-propylamino-5-triazine)—on the induction of nitrate reduc–tase and its in vivo activity was studied in detached leaves of Hordeum vulgare L. All increased both extractable nitrate reductase activity and nitrate content. The increases occurred at optimum temperatures for growth and at several concentrations of nitrate. It was also determined that the herbicides did not protect the enzyme against inactivation in vivo. Although the extractable nitrate reductase was greater, the in vivo activity of nitrate reductase was decreased in the presence of the herbicides resulting in a higher internal concentration of nitrate. Since in viva nitrate reduction is dependent upon photosynthesis it is reasonable that reduction is decreased by these known inhibitors of photosynthesis. Hence, the effect of the inhibitors on induction of nitrate reductase activity may be secondary. The higher concentration of nitrate resulting from a decreased rate of in vivo reduction in the presence of the inhibitors could conceivably be responsible for the greater corutent of nitrate reductase.     

PREPARATION AND CHARACTERIZATION OF PROTOPLASTS OBTAINED FROM THE PRASINOPHYTE SCHERFFELIA DUBIA (CHLOROPHYTA)1

The prasinophyte genera Scherffelia and Tetraselmis are the only genera that form a cell wall by an extracellular fusion of scales called a theca. We established a protocol for the production of protoplasts from Scherffelia dubia Pascher emend. Melkonian et Preisig using 3 mM Ellman's reagent (5,5′‐dithio‐bis‐2‐nitrobenozoic acid [DTNB]). Protoplasts analyzed by EM lacked flagella and thecae but were otherwise similar to control cells. In response to treatment with DTNB, many protoplasts synthesized new thecal scales in the Golgi apparatus, indicating that cells attempted to regenerate new cell walls. However, complete regeneration of the thecae only occurred once DTNB was washed out from the medium. At higher DTNB concentrations (5 mM), two protoplasts were found within the parental cell wall and scales accumulated between the plasma membrane of the protoplasts and the original theca but failed to form a new theca.     

Triterpenes from Douglas fir sapwood

The saponified ether-soluble extractives of Douglas fir sapwood contained (24R)- 4α,14α,24-trimethyl-9β,19-cyclo-5α-cholestan-3β-ol(24R-cyclocucalanol),a new natural product; 4α,14α-dimethyl-9β,19-cyclo-24-methylene-5α-cholestan-3β- ol (cycloeucalenol); and (24R)-4α,24-dimethyl-5α-cholest-7-en-3β-ol (24R- methyllophenol); this is the first time they have been reported from Douglas fir.     

Sensitive high-performance liquid chromatographic assay for motexafin gadolinium and motexafin lutetium in human plasma

We present new HPLC methods for the quantitation in human plasma of two investigative metallotexaphyrin agents, motexafin gadolinium (Gd-Tex) and motexafin lutetium (Lu-Tex). Each assay uses: the other texaphyrin analogue as an internal standard; protein precipitation with acetonitrile:methanol (50:50, v/v); an ODS reversed-phase column; an isocratic mobile phase of 100 mM ammonium acetate, pH 4.3:acetonitrile:methanol (59:21:20, v/v/v); and absorbance detection at 470 nm. The Gd-Tex assay has a lower limit of quantitation (LLOQ) of 0.01 μM and is linear between 0.01and 30 μM. The Lu-Tex assay has an LLOQ of 0.1 μM and is linear between 0.1 and 30 μM. The assays are suited for in vivo preclinical studies and clinical trials because they require minimal amounts of plasma, are sensitive, and involve a 30-min run time. These assays are important tools for evaluating the potential of Gd-Tex and Lu-Tex as a radiation enhancer and photosensitizer, respectively.     

Studies on the Syntheses of the Pyrethrin Analogues and their Biological Activities

Some new allethronyl esters of cyclopropanecarboxylic acids were prepared, and their insecticidal activities were tested against common housefly. Allethronyl esters of cyclopropanecarboxylic acids having 3,4-methylenedioxyphenyl group on the cyclopropane ring were more or less toxic. Among them, allethronyl 2,2-dimethyl-3-(3′,4′-methylenedioxyphenyl)-cycloopropane-l-carboxylate was found to be more toxic than α-dl-trans-allethrin, and the calculated relative effectiveness were 1.48 and 1.21 on the mortality and knock-down activity respectively as compared with α-dl-trans-allethrin.     

Freezing damage and frost tolerance of the photosynthetic apparatus studied with isolated mesophyll protoplasts of Valerianella locusta L.

Mesophyll protoplasts were isolated from unhardened and cold-acclimated leaves of Valerianella locusta L. and subjected to freeze-thaw treatment. To evaluate the extent and course of freezing injury, photosynthetic reactions of whole protoplasts and of free thylakoid membranes, liberated from protoplasts by osmotic lysis, were measured. In addition, the integrity of the protoplasts was determined by microscopy. The results reveal an increased frost tolerance of protoplasts isolated from acclimated leaves with respect to all parameters measured. CO₂-dependent O₂ evolution (representing net photosynthetic CO₂ fixation of protoplasts) was the most freezing-sensitive reaction; its inhibition due to freeze-thaw treatment of protoplasts was neither correlated with disintegration of the plasma membrane, nor was it initiated by inactivation of the thylakoid membranes. The frost-induced decline of protoplast integrity was not closely correlated to thylakoid damage either. Freezing injury of the thylakoid membranes was manifested by inhibition of photosynthetic electron transport and photophosphorylation. Both photosystems were affected by freezing and thawing with strongest inhibition occurring in the water-oxidation system or at the oxidizing site of photosystem II. Photophosphorylation responded more sensitively to freezing stress than electron transport, although uncoupling (increased permeability of the thylakoid membranes to protons) was not a conspicuous effect. The data are discussed in relation to freezing injury in leaves and seem to indicate that frost damage in vivo is initiated at multiple sites.Abbreviations Chl chlorphyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCIP 2,6-dichlorophenolindophenol - DPC 1,5-diphenylcarbazide - Hepes 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid - MES 2-(N-morpholino)-ethanesulfonic acid - PS I photosystem I - PS II photosystem II     

Ethylene formation in Pisum sativum and Vicia faba protoplasts

Protoplasts isolated from leaves of peas (Pisum sativum L.) and of Vicia faba L. produced 1-aminocyclopropane-1-carboxylic acid (ACC) from endogenous substrate. Synthesis of ACC and conversion of ACC to ethylene was promoted by light and inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and carbonyl cyanide m-chlorophenylhydrazone. Aminoethoxyvinylglycine inhibited ethylene synthesis to a minor extent when given during incubation of the protoplasts but was very effective when added both to the medium in which the protoplasts were isolated and to the incubation medium as well. Radioactivity from [U-¹⁴C]methionine was incorporated into ACC and ethylene. However, the specific radioactivity of the C-2 and C-3 atoms of ACC, from which ethylene is formed, increased much faster than the specific radioactivity of ethylene. It appears that ACC and ethylene are synthesized in different compartments of the cell and that protoplasts constitute a suitable system to study this compartmentation.Abbreviations ACC 1-Aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - CCCP carbonyl cyanide m-chlorophenylhydrazone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea