The Metabolism of Abscisic Acid

The light-catalysed isomerization of (+)-abscisic acid (ABA)to its trans isomer during isolation from leaves was monitoredby the addition of (±)-[2-¹⁴C]ABA to the extraction medium.(+)Trans-abscisic acid (t-ABA) was found to occur naturallyin rose (Rosa arvensis) leaves at 20µg/kg fresh weight;(+)-ABA was present at 594µg/kg. (±)-[2-¹⁴D]Trans-abscisicacid was not isomerized enzymically to ABA in tomato shoots. (±)-Abscisic acid was converted by tomato shoots to awater-soluble neutral product, ‘Metabolite B’, whichwas identified as abscisyl-ß-D-glucopyranoside. When(±)-[2-¹⁴C]trans-abscisic acid in an equimolar mixturewith (±)-[2-¹⁴C}ABA was fed to tomato shoots it was convertedto its glucose ester 10 times faster than was ABA. Trans-abscisyl-ß-D-glucopyrano8ide only was formedfrom (±)-[2-¹⁴C]t-ABA in experiments lasting up to 30h. Glucosyl abscisate was formed slowly from ABA and the freeacid fraction contained an excess of the unnatural (–).ABAas did the ABA released from abscisyl-ß-D-glucopyranosideby alkaline hydrolysis. The (+).ABA appeared to be the solesource of the acidic ‘Metabolite C" previously noted. The concentrations of endogenous (+)-, (+)-[2-¹⁴C]-, and (–)-[2-¹⁴C]ABAremaining as free acid, and also in the hydrolysate of abscisyl-ß-D-glucopyranoside,were measured by the ORD, UV absorption, and scintillation spectrometryof highly purified extracts of ABA from tomato shoots whichhad been supplied with racemic [2-^l4C]ABA.     

Characterization of an abscisic acid carrier in suspension-cultured barley cells

The uptake of [³H]-abscisic acid in barley (Hordeum vulgareL. cv. Heartland) cell cultures was found to be mediated throughboth non-saturable and saturable components. The kinetic parametersof the saturable component, determined at pH 4.5 and 21 °C,showed a K_m for natural or (+ )-ABA of 1.3±0.7µMand an apparent V_mex of 7.0 ± 2.8 nmol g^–1 cellsh^–1. The carrier showed a strong preference for the naturalenantiomer of ABA as compared to the unnatural one. Other substancestested, e.g. amino acids, organic acids, and other growth regulators,did not appear to interfere with the carrier-mediated uptakeof ABA. At low external concentrations of ABA (below 2.0 µM),the saturable component was greater than the diffusion component.Similarly, between pH 4.0 and 6.0, the saturable uptake wasresponsible for more than 50% of the total uptake. The carriermay be important in vivo for mediating uptake when endogenouslevels of ABA are low (c. 1 µM). The carrier specificity was evident in inhibition experimentsdone with ABA analogues. Our data showed that the carrier couldaccommodate small modifications in the ABA structure. Four analogueswere able to compete efficiently with ( + )-ABA for the bindingsite of the carrier. Three of these competitors were of the(+)-series. Only one ( –)-analogue, (–)-ABA, wasable to inhibit markedly the saturable uptake of ( + )-ABA.The induction of the ABA-respons-ive gene WCS120 (Houde et al.,1992) presented stricter requirements for the ABA molecule thanthe carrier, although with a similar preference for the ( +)-analogues. Besides ( + )-ABA itself, only two of the analoguestested, both ( + )-series, were able to induce the WCS120 geneafter a 24 h incubation period. The absence of correlation betweenthe activity of the analogues as ABA inhibitors in the carriersystem, and their capacity to induce the WCS120 gene tend tosuggest that the carrier is not directly involved in the signaltransduction pathway leading to the induction of this specificgene. Key words: Abscisic acid, barley, gene induction, Hordeum vulgare, uptake carrier     

Validation of a radioimmunoassay for (+)-abscisic acid in extracts of apple and sweet-pepper tissue using high-pressure liquid chromatography and combined gas chromatography-mass spectrometry

A radioimmunoassay for (+)-abscisic acid (ABA) was developed and applied to the analysis of free ABA in extracts of apple (Malus pumila Mill.) and sweet pepper (Capsicum annuum L.) leaves at various stages during extract purification. Conjugates of ABA, were quantified after alkaline hydrolysis. The validity of the radioimmunoassay was tested by comparison of immunoassay estimates of ABA at different levels of extract purity with high-pressure liquid chromatography (HPLC) and combined gas chromatography-mass spectrometry. The antiserum, raised against (+)-ABA, was almost equally sensitive to (-)-ABA. Serum cross-reactivity with the methyl ester of ABA was 160% and with the glycosyl ester of ABA was 34%. Cross-reactivity with protein-ABA conjugates was very slight for C₄-conjugated keyholelimpet haemocyanin, but about 1000% for C₁-conjugated alkaline phosphatase. Other compounds tested showed extremely low or undetectable cross-reactivities. Further evidence for the specificity of the assay came from the agreement between the results using different assay methods for both apple and pepper extracts, and from the observation that the only zone of immunoreactivity on HPLC elution profiles corresponded with authentic (+)-ABA. The use of polyvinylpyrrolidone in the assay minimised interference by other substances in plant extracts. In pepper, free ABA levels increased rapidly during water stress and recovered to pre-stress levels within two days after rewatering. Levels of ABA conjugates were significantly lowr than free ABA in unstressed plants, and also increased rapidly with stress, although not to the same extent as free ABA, and did not recover as rapidly as did free ABA. In apple, levels of free ABA and of ABA conjugates both increased more than twofold over a two-week period of water stress. In contrast to pepper, however, immunoreactivity of the conjugate fraction was increased by hydrolysis, indicating that different ABA conjugates predominate in the two species.Abbreviations ABA abscisic acid - GC-MS combined gas chromatography-mass spectrometry - HPLC high-pressure liquid chromatography - Me-ABA methyl ester of ABA - PVP polyvinylpyrrolidone - RIA radioimmunoassay     

2-Trans-Abscisic Acid Biosynthesis and Metabolism of ABA-Aldehyde and Xanthoxin in Wild Type and the aba Mutant of Arabidopsis thaliana

Abscisic acid (ABA) and 2-trans-ABA (t-ABA) biosynthesis werestudied in wild type Landsberg erecta and the three allelicaba mutants of Arabidopsis thaliana (L.) Heynh., which are impairedin epoxy-carotenoid biosynthesis. Labelling experiments with¹⁸O₂and mass spectrometric analysis of [¹⁸O]ABA and its catabolitesABA-glucose ester (ABA-GE) and phaseic acid (PA), and t- ABAand t-ABA-GE, showed that t-ABA biosynthesis was less affectedthan ABA biosynthesis by mutations at the ABA locus. The aba-4allele caused the most severe impairment of ABA biosynthesiscompared with the other two mutant alleles aba-1 and aba-3,yet aba-4 plants synthesized as much t-ABA as wild type Landsbergerecta plants. Feeding experiments with RS- [²H₆]ABA-aldehydeisomers and unlabelled xanthoxin isomers suggest that t-xanthoxinand t-ABA-aldehyde are precursors to ABA and t-ABA in Arabidopsis Key words: ABA-alcohol, ABA-aldehyde, ABA-glucose ester, ¹⁸O₂ labelling, phaseic acid     

Endogenous cis,trans-Abscisic Acid and Pea Seed Development: Evidence for a Role in Seed Growth from Changes Induced by Temperature

Measurements were made using GC/MS SIM¹ of the effects of temperatureon cis,trans-ABA levels in developing ovules and embryos oftwo pea genotypes contrasted in seed size. These effects werethen related to differences in the growth of the pods, seeds,embryos, and testae. In both genotypes high temperatures hastenedthe onset and rate of logarithmic and then linear growth, greatlyshortening the duration of pod and seed development but withoutgreatly altering seed size. Cis,trans-ABA was most concentratedxin the ovules immediately after fertilization. It also accumulatedin the embryo, more rapidly in the larger-seeded line, duringseed maturation. The stage when accumulation in the embryo beganwas the same irrespective of temperature. Accumulation ceasedwhen the pods started to desiccate. The effects of differentconstant temperatures on the maximum levels of embryo cis,trans-ABAwere relatively small and confounded in one genotype by variationin ovule abortion and in the other by differences in the stagewhen cis,trans-ABA accumulation ceased. However, when plantswere transferred from 13 °C to 29 °C at two differentstages during seed maturation, further seed growth was greatlyinhibited coincident with a substantial increase in embryo cis-trans-ABA.The results suggested a role for cis,trans-ABA in the controlof cotyledon enlargement during the linear phase of seed growth.     

The Resolution by HPLC of RS-[2-14C]Me 1', 4'-cis-Diol of Abscisic acid and the Metabolism of (-)-R- and (+)-S-Abscisic Acid

The R- and S-enantiomers of racemic [2-¹⁴C]Me 1', 4'-cis-diolof abscisic acid have been separated by high performance liquidchromatography on an optically-active Pirkle column. R-[2-¹⁴C]-and S-[2-¹⁴C]abscisic acids, formed from the Me 1', 4'-cis-diolby oxidation and alkyline hydrolysis were fed to tomato shootsand the extracts analysed by reversed phase high performanceliquid chromatography. R-[2-¹⁴C]abscisic acid formed mainlythe abscisic acid glucose ester (ABAGE), abscisic acid l'-glucoside(ABAGS) and an uncharacterized conjugate. Dihydrophaseic acid4'-B-D-glucoside, the major metabolite of RS-abscisic acid intomato shoots, was found to be derived virtually exclusivelyfrom the natural, S-abscisic acid. Phaseic acid and conjugatesof abscisic acid were also found as products of the naturallyoccurring enantiomer. The resolution method was used to measurethe relative proportions of R and S enantiomers in the freeacid liberated from conjugates formed from RS-[2-¹⁴C]ABA fedto shoots. The ratios show an excess of the R-enantiomer: 5.8:1, ABAGE; 29.4: 1, ABAGE; 8.3: 1 for an uncharacterized conjugateand 6.1: 1 for the residual free [2-¹⁴C]ABA. Key words: ABA, HPLC, Tomato     

Synthesis of an S-(2-Aminoethyl)-L-Cysteine Conjugate in S-(2-Aminoethyl)-L-Cysteine- Resistant Adenine-Auxotrophic Cells of Datura innoxia Mill

A line of S-(2-aminoethyl)-L-cysteine-resistant adenine-auxotrophiccells (Ad^–AEC^r strain) was isolated from adenine-auxotrophiccells (Ad^– strain) of Datura innoxia Mill by a stepwiseselection method. Ad^–AEC^r and Bl cells, which were clonedfrom the original Ad^–AEC^r cells, were able to grow activelyon medium that contained 10 mM S-(2-aminoethyl)-L-cysteine (AEC),whereas the growth of Ad^– cells ceased completely in thepresence of 0.5 mM AEC. The resistant phenotype has been maintainedfor at least 10 months in culture on medium without AEC. Levels of free lysine in Ad^–AEC^r and Bl cells were similarto that in Ad^– cells. By contrast, the level of free AECin Ad^–AEC cells was 10-fold lower than in Ad^– cellsand no free AEC was detectable in Bl cells. However, acid hydrolysisof extracts from Ad^–AEC^r and Bl cells resulted in a remarkableincrease in levels of detectable AEC. This result indicatesthat conjugated AEC is synthesized and accumulated in the AEC-resistantcells. The level of the AEC conjugate in Bl cells increasedwith increases in the concentration of AEC in the culture medium,while intracellular levels of AEC were so low as not to be detectablein the case of cells grown on medium supplemented with AEC atless than 1 mM. The AEC conjugate was also detected in Ad^–cells, but at lower levels than in the AEC-resistant cells.In addition, AEC was found to be incorporated into soluble proteinsin Ad^– cells. These results suggest that the resistance of AEC-resistant cellsof Datura innoxia is accomplished via acceleration of the synthesisof the AEC conjugate which prevents any increase in intracellularlevels of free AEC. ¹Present address: Institute for Biology and Chemistry, TsumuraCo.Ltd., Inashiki, Ibaraki, 300-03 Japan. ²Present address: North Kanto Shop, Sakata Seed Co. Ltd.,Saitama,347 Japan.     

Types, Formation, and Metabolism of Cytokinins in Leaves of Alnus Glutinosa (L.) Gaertn.

The nature of the cytokinins present in mature, expanded leavesof alder (Alnus glutinosa (L.) Gaertn.) was investigated usinga chromatographic system capable of partially resolving zeatinfrom (?)-dihydrozeatin. A zeatin-like cytokinin, present asboth a ‘free’ form and as a polar conjugate, postulatedto the the O-ß-D-glucoside of zeatin, accounted forthe bulk of the cytokinin activity (detected by the soybeancallus assay). Studies on the fate of [8-¹⁴C]zeatin supplied to detached alderleaves indicated this cytokinin to undergo extensive metabolism.An appreciable proportion of the radioactivity was incorporatedinto 80% methanol-insoluble and soluble acidic/neutral fractions,while adenosine- and adenine-like peaks were prominent metabolitesin the basic n-butanol-soluble fraction. Small amounts of glucosides,with the properties of both zeatin-O-ß-D-glucosideand dihydrozeatin-O-ß-D-glucoside were formed, thelatter becoming the most prominent with time. The ability of alder leaves to form glucoside directly from(?)-dihydrozeatin was assessed using the soybean callus assay.(?)-Dihydrozeatin was subject to a relatively rapid, continuousand substantial conversion to its putative O-ß-D-glucosideand cytokinin activity was consequently conserved while, incontrast, leaves supplied with zeatin exhibited a progressiveloss of cytokinin activity and, in agreement with the radioactivityexperiments, produced only a small amount of activity attributableto the putative O-glucosides. The significance of the observed cytokinin metabolism is discussedin relation to the endogenous cytokinin status of the leaf.     

The Role of Glutamate Decarboxylase and {gamma}-Aminobutyric Acid in Germinating Barley

The variations in glutamate decarboxylase activity and in glutamicacid and -ABA concentration have been measured in barley embryosduring the uptake of water and in the roots and shoots for upto 6 days of growth. Glutamate decarboxylase activity was relativelysteady in the embryos during soaking but rose rapidly once growthbegan. This development paralleled an increase in the concentrationof glutamic acid in both roots and shoots at a time when theconcentration of -ABA was falling. During soaking in aeratedwater, the -ABA content of the embryos rose for 36 h, at whichpoint it accounted for 35 per cent of the soluble amino acids.-ABA was found to be a major free amino acid in roots but notin shoots. Experiments in vivo involving ¹⁴C-labelled glutamicacid and -ABA indicated that carbon from -ABA passed very rapidlyinto the citric-acid cycle intermediates and also that, throughoutthe period studied, -ABA was formed from glutamic acid despitethe alterations in relative concentrations of these amino acidsin the growing tissue.     

Natural Inhibitors in Normal and Dwarf Peas

Extracts of light- and dark-grown, normal and dwarf pea seedlings(Pisum sativum L. cv. Alaska and Radio respectively) were purifiedby solvent partitioning, column, paper and thin layer chromatography.Conventional acid-base partitioning was modified because thelarge volumes of material processed caused considerable crosscontamination between neutral and acidic phases. At each stepof the purification, fractions were tested for inhibitory activitywith the wheat coleoptile and pea section tests. Recovery ofabscisic acid was monitored using ¹⁴C-abscisic acid. Estimatesof abscisic acid content were marie using gas-liquid chromatographyand the wheat coleoptile bioassay. Two main inhibitors were found; one of these was identifiedas (+)-abscisic acid, the other (inhibitor Y) has not been identifiedbut displays chromatographic properties which suggest that itis neutral in nature. Abscisic acid was found in both rootsand shoots of light- and dark-grown pea seedlings. InhibitorY was found in trace amounts in the roots of dark-grown plantsbut could not be detected in the shoots. Growth in light induceda manifold increase in inhibitor Y concentration compared withdark-grown plants. The level of Y was threefold greater in light-growndwarf shoots than in comparable light-grown tall shoots. Therewas, thus, a correlation between the concentration of inhibitorY and the light-induced inhibition of stem elongation.     

Phytochemical studies on tobacco alkaloids XIV. The occurrence and properties of putrescine N-methyltransferase in tobacco roots

Putrescine N-methyltransferase, a new enzyme catalyzing theformation of N-methylputrescine from putrescine and S-adenosyl-L-methioninewas found in roots of tobacco plants. The enzyme was purified30-fold from crude extracts of tobacco roots. NMethylputrescinewas identified as the reaction product by comparison with theauthentic compound. The enzyme had a pH optimum between pH 8and 9, and a molecular weight of about 60,000, as determinedby gel filtration. Km values for putrescine and 5-adenosyl-L-methioninewere 4.0 x 10^–4 M and 1.1 x 10^–4 M, respectively.Enzyme activity was inhibited by N-chloromercuribenzoate andAg⁺. No cofactors were required. Of the various substrates tested,only putrescine served as a methyl acceptor. The enzyme waslocalized exclusively in the roots and its activity was greadyenhanced by decapitation. The presence of putrescine N-methyltransferase in tobacco rootsstrongly suggests that N-methylputrescine participates as anintermediate in nicotine biosynthesis. (Received March 2, 1971; )     

Intracellular pH Regulation in an Acidophilic Unicellular Alga, Cyanidium caldarium: 31P-NMR Determination of Intracellular pH

The intracellular pH of an acidophilic unicellular alga, Cyanidiumcaldarium, was determined as a function of external pH by ³¹Pnuclear magnetic resonance. The algal cells incubated underaerobic conditions or under anaerobic and illuminated conditionsmaintained the intracellular pH in the range from 6.8 to 7.0even when the external pH was changed from 1.2 to 8.4. Underanaerobic and dark conditions, however, the intracellular pHacidified at the acidic pH region of the external medium. Theacidified intracellular pH reversibly returned to neutral eitheron aeration or illumination. The results indicate that, in Cyanidiumcells growing in extremely acidic environments, an active H⁺efflux (H⁺ pump) which depends on metabolic activity (respirationor photosynthesis) is essential to maintain the intracellularpH at a constant physiological level against the passive H⁺leakage due to the steep pH gradient across the cell membrane. (Received March 19, 1986; Accepted July 17, 1986)     

Endogenous gibberellins and inhibitors in caryopses of rye

Gibberellins A₈, A₁₆, A₂₄, and abscisic acid were identified by GC-MS of derivatized extracts from immature fruits of Secale cereale. Mature caryopses contained ABA and trans-ABA in a ratio 1:1 as well as 4′-dihydrophaseic acid. During milk ripeness a neutral GA conjugate was detected. Free GA, afforded by enzymatic hydrolysis of the conjugate, was chromatographically identified as GA₁₆     

Synthesis of L(+) Tartaric Acid from 5-Keto-D-gluconic Acid in Pelargonium

5-Keto-D-[1-¹⁴C]gluconic acid, the most effective precursorof L(+)tartaric acid among all labeled compounds which haveever been tested in grapes, was found to be a good precursorof L(+)tartaric acid in a species of Pelargonium. The synthesisof labeled L(+)tartaric acid from D-[1-¹⁴C]glucose in Pelargoniumwas remarkably depressed when a 0.5% solution of D-gluconateor 5-keto-D-gluconate was administered continuously to leavestogether with D-[1-¹⁴C]glucose. Our results provide strong evidence that D-[1-¹⁴C]glucose ismetabolized in Pelargonium to give labeled L(+)tartaric acidvia (probably D-gluconic acid and) 5-keto-D-gluconic acid withoutpassing through L-ascorbic acid. Labeled L-idonic acid was found in young leaves of Pelargoniumwhich had been labeled with L-[U-¹⁴C]ascorbic acid. The synthesisof the labeled L-idonic acid increased when a 0.1% solutionof L-threonate was administered continuously to leaves togetherwith L-[U-¹⁴C]ascorbic acid. Specifically labeled compounds, recognized as the members ofthe synthetic pathway for L(+)tartaric acid from L-ascorbicacid via L-idonic acid in grapes, were administered to youngleaves of Pelargonium. Each compound (2-keto-L-[U-¹⁴C]idonicacid, L-[U-¹⁴C]idonic acid, 5-keto-D-[1-¹⁴C]gluconic acid and5-keto-D-[6-¹⁴C]gluconic acid) was partly metabolized, as ingrapes. The metabolic pathway starting from L-ascorbic acidto L(+)tartaric acid via L-idonic acid, however, did not actuallycontribute to the synthesis of L(+)tartaric acid in Pelargoniumprobably because the activity of each metabolic step was muchlower than that observed in grapes. (Received May 28, 1984; Accepted July 30, 1984)     

Metabolism of S-methylcysteine and pectin esterification in Chinese cabbage, Brassica pekinensis Rupr.

S-Methyl-L-cysteine was actively metabolized in Chinese cabbageand carbon from its methyl group was distributed into both thesoluble and insoluble fractions. The high incorporation of ¹⁴Cfrom the methyl group into the insoluble fraction after administeringof S-methyl-L-cysteine-¹⁴CH₃, and our previous results thatS-methyl-L-cysteine is demethylated to give cysteine, suggestthat S-methyl-L-cysteine might act as a methyl donor in Chinesecabbage. To obtain evidence for this possibility, incorporationof the methyl-¹⁴C of S-methyl-L-cysteine into methyl estersof pectic substances was investigated. Most of the ¹⁴C incorporatedinto pectic substances was liberated by treatment with dilutealkali and pectin esterase. The results show that S-methyl-L-cysteineacts as a methyl donor to form pectin ester. (Received October 12, 1971; )     

The Effects of Leaf Age and Senescence on the Distribution of Carbon in Lolium temulentum

Assimilate distribution in leaves of Lolium temulentum was establishedby root absorption of [¹⁴C]sucrose and after exposure to ¹⁴CO₂.Age determined the amount of carbon assimilated, with more labelbeing incorporated during expansion than at maturity. Duringsenescence ¹⁴C assimilation was much lower. Ethanol-solubleextracts from various tissues of root-labelled plants containedmost of the radioactivity chiefly in basic and acidic compounds.The neutral fraction was composed predominantly of sucrose. Sucrose was comparably labelled in leaves from plants fed equalamounts of either [¹⁴C]sucrose, glucose, or fructose and onlytraces of labelled monosaccharides appeared in extracts. Radioactive sucrose was translocated rapidly from mature leaveswhereas, in the expanding leaf, carbon incorporation was directedtowards growth and the greater proportion of label present atligule formation was in ethanol-insoluble material. Induced senescence, of a mature leaf fed during expansion, produceda rapid loss from the pool of insoluble ¹⁴C. This was accompaniedby a reduction in the contents of chlorophyll and soluble proteinand an accumulation of amino acids. The onset of senescencecaused changes in leaf sugar levels which were correlated withincreased rates of respiration.     

Conversion of (+)-Dihydroquercetin to 3,4-cis-Leucocyanidin by a Reductase Extracted from Cell Suspension Cultures of Cryptomeria japonica

A soluble, NADPH-dependent reductase catalyzing the reductionof (+)-dihydroquercetin to 3,4-cis-leucocyanidin (5,7,3',4'-tetrahydroxyflavan-3,4-cis-diol)was demonstrated in an enzyme preparation from a cell suspensionculture of Japanese cedar (Cryptomeria japonica D. Don). TheK_m value for (+)-dihydroquercetin was 48µM. The enzyme,which was purified 26.2-fold, could also catalyze the reductionof (+)-dihydrokaempferol to 3,4-cis-leucopelargonidin (5,7,4'-trihydroxyflavan-3,4-cis-diol). The enzyme had a pH optimumof 7 and a molecular weight of 133,000. It was inhibited byCu²⁺ and iodoacetate, but not by p-chloromercuribenzoate. Duringthe growth stages of the cell suspension cultures, an increasein reductase activity proceeds an increase in procyanidin content,as might be expected. (Received November 25, 1987; Accepted April 11, 1988)     

INVESTIGATION OF THE ACIDIC ALLOMONE OF THE GASTROPOD MOLLUSC PHILINE APERTA BY MEANS OF ION CHROMATOGRAPHY AND HISTOCHEMICAL LOCALISATION OF SULPHATE AND CHLORIDE IONS

Improved methods of collection of acidic allomones from theskin of the gastropod mollusc Philine aperta minimised contaminationby the anions of sea water and of blood. Neat allomone extractsremained stable in the laboratory for up to 15 months at 5–8°C,having a pH value of 2–3. Diluted extracts (1: 9 or 1:99) gradually lost their activity. Analyses by the Dionex H.P.ion chromatography apparatus showed that organic ions were presentonly in trace quantities in the allomone samples, and the principalinorganic anions were chloride and sulphate (their concentrationsapproached parity in the purest preparations). Measurementsof Cl^– varied in different samples from 2062 to 3989 p.p.million, and of SO₄^2–from 1272 to 3152 p.p. million. TheSO₄^2- CI^- ratios varied from 0.62 to 0.84. New observations are presented on the rate of in situ precipitationof BaSO₄ in preparations of the skin of Philine. The rate ofpenetration of BaCl₂ into the skin was approximately linear,even when the animal had become moribund (30 min). BaSO₄ crystalgrowth increased during the first 30 min of exposure, but laterdeclined, for reasons that are not clear. The method yieldedclear outlines of the acid sacs in histological preparations. A silver nitrate irrigation method was devised in order to investigatechloride ions in the acidic epithelium of Philine. The advanceof AgNO₃ into the tissue was usually smooth-fronted, but siteswere evident where the acid sacs had acted as channels for penetration.The silver deposits took the form of uniform small particles,often precipitated against the inner wall of an acid sac, butsometimes forming a central cluster. The method was of valuein building up a picture of structural relations as well asthe chemical composition of the acid sacs of Philine (Received 18 February 1985;     

Phenolic Growth Inhibitors Isolated from Dormant Buds of Sugar Maple (Acer saccharum Marsh)

Phenolic growth-inhibiting substances were isolated from dormantbuds of sugar maple (Acer saccharurn Marsh) by paper chromatographyof their aqueous methanolic extracts. Inhibition was determinedin germination bioassays using lettuce (Lactuca sativa var.Grand Rapids) seeds. An inhibition of some 20% was attributedto four major phenolics identified as ferulic, vanillic, p-coumaric,and caffeic acids. Quercetm was also present and caused a weakstimulation of growth. Fractionation of the extract with acidic, neutral, and basicgroups indicated that more phenolic compounds were in the acidicgroup than in the neutral, while none were in the basic group. On a seasonal basis, autumn buds had a higher phenolic contentthan winter and spring buds. The results presented are not inconsistentwith the view that growth-inhibitory phenolic compounds couldcontribute to dormancy in sugar maple buds.