Retention of Metabolic and Differentiation Potentials of Green Lavandula vera Callus after Freeze-Preservation

Green Lavandula vera callus that produced biotin was preservedsuccessfully in liquid nitrogen for up to 3 weeks. We foundthat the callus recovered after the freeze-preservation, retainingnot only the biosynthetic capability for biotin but also differentiationpotentials such as chloroplast development and plantlet formation.The significance of retention of the metabolic and differentiationpotentials of the callus is discussed in terms of the freeze-preservationof plant genetic resources. ^* This study is dedicated to the late Professor J. Ashida. (Received September 1, 1982; Accepted November 5, 1982)     

Biotin uptake by human colonic epithelial NCM460 cells: a carrier-mediated process shared with pantothenic acid

Previous studies showed that the normal microflora of the largeintestine synthesizes biotin and that the colon is capable of absorbingintraluminally introduced free biotin. Nothing, however, is known aboutthe mechanism of biotin absorption in the large intestine and itsregulation. To address these issues, we used the human-derived,nontransformed colonic epithelial cell line NCM460. Theinitial rate of biotin uptake was found to be1) temperature and energy dependent,2)Na⁺ dependent (coupling ratio of1:1), 3) saturable as a function ofconcentration [apparent Michaelis constant(K_m) of 19.7 µM], 4) inhibited bystructural analogs with a free carboxyl group at the valeric acidmoiety, and 5) competitivelyinhibited by the vitamin pantothenic acid (inhibitionconstant of 14.4 µM). Pretreatment with the protein kinase C (PKC)activators phorbol 12-myristate 13-acetate (PMA) and1,2-dioctanoyl-sn-glycerolsignificantly inhibited biotin uptake. In contrast, pretreatment withthe PKC inhibitors staurosporine and chelerythrine led to a slight, but significant, increase in biotin uptake. The effect of PMA was mediatedvia a marked decrease in maximal uptake velocity and aslight increase in apparentK_m. Pretreatmentof cells with modulators of the protein kinase A-mediated pathway, onthe other hand, showed no significant effect on biotin uptake. Theseresults demonstrate, for the first time, the functional existence of aNa⁺-dependent, specializedcarrier-mediated system for biotin uptake in colonic epithelial cells.This system is shared with pantothenic acid and appears to be under theregulation of an intracellular PKC-mediated pathway.

     

Comparison of Free and Bound Amino Acids in White and Green Tissues of Variegated Tobacco Plants

Qualitative and quantitative comparisons of free and bound aminoacids and soluble proteins in white and green tissues of variegatedtobacco leaves were made. White tissue contained more free andless bound amino acids than green tissue, although the sum ofthe total amino acids did not differ significantly between thetwo tissues. The major free amino acids in white tissue wereglutamine and asparagine, whereas those in green tissue wereglutamic acid, aspartic acid and -aminobutyric acid. The contentsof fraction 1 protein and 70 S ribosomes in white tissue werenegligible in comparison with those found in green tissue, butthe amounts of other soluble protein components and the 80 Sribosomes were at the same level in both tissues. (Received October 21, 1981; Accepted January 28, 1982)     

The selection of cultured plant cell lines producing high levels of biotin

We have found that biotin is synthesized in many species of cultured plant cells, e.g. Lavandula vera Labiatae), Nicotiana tabacum (Solanaceae) and Glycine max Leguminosae). Cultured green L. vera cells grown under light contained the greatest amounts of free biotin of the cells studied although the specific amounts varied among the cell lines. Cell lines were selected after their free biotin contents had been analysed. Cells containing large amounts of free biotin were cultured repeatedly, analysed and reselected. Lines with high levels of free biotin were obtained from cells which survived on a medium containing pimelic acid and l-alanine or from gamma irradiated cells. One L. vera cell line obtained from irradiated cells contained seven times the amount of free biotin found in the original unselected cultured cells and four and a half times that found in the leaves.     

Changes in the Levels of Free Amino Acids in Various Regions of Cuscuta during Growth

The angiospermous plant parasite Cuscuta derives reduced carbonand nitrogen compounds primarily from its host. Free amino acidsalong Cuscuta vines in three zones, viz., 0 to 5 cm, 5 to 15cm, and 15 to 30 cm, which in a broad sense represent the regionof cell division, cell elongation and differentiation and vasculartissue differentiation respectively, were quantitatively estimated.The free amino acid content was the highest in the 0 to 5 cmregion and progressively decreased along the posterior regionsof the vine. The haustorial region showed the lowest contentof free amino acids. In general, the free amino acid contentin samples collected at 7 p.m. was found to be higher than thatin the samples collected at 7 a.m. Three basic amino acids,histidine, the uncommon amino acid -hydroxyarginine, and arginineconstituted more than 50% of the total free amino acids in allthe zones studied except the haustorial region. Aspartic acidand glutamic acid constituted the major portion in the acidicand neutral fraction of amino acids. Glutamine, asparagine,threonine, and serine were eluted together and occurred in substantialamounts. -Hydroxyarginine constituted the largest fraction inthe cut end exudate of Cuscuta and presumably appeared to bethe major form of transport amino acid. -Hydroxyarginine wasalso a major constituent of the basic amino acids in Cuscutavines parasitizing host plants from widely separated families,suggesting that this amino acid is a biosynthetic product ofthe parasite rather than that of the hosts. Also, U-¹⁴C argininewas converted to -hydroxyarginine by cut Cuscuta vines, suggestingthat -hydroxyarginine is synthesized de novo from arginine byCuscuta. (Received March 30, 1987; Accepted June 7, 1988)     

Nitrogenous Compounds and Nitrogen Metabolism in the Liliaceae: II. The Nitrogenous Compounds of Leaves of the Genus Tulipa: Environmental Effects on the Composition of Tulipa gesneriana

Because of the predominance of -methyleneglutamine in Tulipagesneriana a quantitative, chromatographic investigation hasbeen made to show the factors which determine the compositionof the alcohol-soluble nitrogen fraction in Tulipa A comparative study of the amino-acids that occur free in theleaves of fourteen different species of Tulipa has been made,-methyleneglutamine varied from as much as 60–70 per cent,of the amino-acid nitrogen down to levels below those whichpermit its chromatographic detection. The effects of temperature during growth and of diurnal variationon the nitrogenous compounds of the leaves of T. getnerianaare described. Excised shoots of T. gesneriana were exposed to light and darknessto affect the balance of protein synthesis and breakdown sothat the concomitant effects on the -methyleneglutamine acidcompounds and other main components of the soluble nitrogenfraction could be determined. Lastly, the relative ease of entry of C¹⁴ from C¹⁴O₂ into thesugars and certain other compounds of T. gesneriana is contrastedwith the relative difficulty with which it enters the -methyleneglutamineof the mature leaves on excised shoots. The occurrence and metabolism of -methyleneglutamine in T. gesnerianaleaves may, therefore, be regarded as additional to and superimposedupon an otherwise typical pattern of nitrogen metabolism: inthese leaves -methylene-glutamine does not assume the role usuallydischarged by glutamine.     

BIOTIN LIBERATION BY THE LICHEN ALGA COCCOMYXA SP. AND BY CHLORELLA PYRENOIDOSA

The unicellular green alga Coccomyxa, a component of the lichenPeltigera aphthosa, liberated about 7.2mµg biotin permg dry weight of cells into the culture medium during a growthperiod of 15–20 days. The corresponding figure for thefree-living alga Chlorella pyrenoidosa was 0.45mµg ofbiotin. Chromatographic analysis indicated that this was freebiotin and not a bound form of the vitamin. The biotin concentrationof rinsed Coccomyxa cells was 1.88mµg per mg dry weightof cells, of which less than 0.01mµg was extractable byhot water. Cells of Chlorella contained 0.16mµg of biotinper mg dry weight, of which 0.11mµg was extractable byhot water. The biotin content of Coccomyxa, which was about12 times that of Chlorella, is thus almost entirely in the boundform. The importance of biotin in the symbiotic interactionsbetween the alga and the fungus in Peltigera is discussed. ¹Present address: University Department of Agriculture, Oxford,England. ²Present address: Institute of Marine Resources, Universityof California, La Jolla, California, U.S.A.     

The sodium pump glutaconyl-CoA decarboxylase from Acidaminococcus fermentans. Specific cleavage by n-alkanols

Glutaconyl-CoA decarboxylase from Acidaminococcus fermentans was inactivated by incubation with n-alkanols at 37 degrees C. The concentration of the alcohol required for complete inactivation decreased with increasing chain length; e.g. 2 M ethanol was as potent as 2 mM hexanol or 0.5 mM decanol. The data indicate a binding of the alcohol to the enzyme with an energy of about 4 kJ/methylene group. Sodium ions prevented the inactivation (50% at 30 mM NaCl). K+, NH4+, Cs+ and Mg2+ had no influence, whereas Li+ was ten times less effective than Na+. The enzyme was cleaved during the inactivation into a soluble part, consisting of the alpha (Mr 120,000) and beta polypeptide chains (60,000), whereas the hydrophobic gamma chain (30,000) precipitated. The soluble part catalysed the sodium-ion-independent but avidin-sensitive glutaconyl-CoA/crotonyl-CoA exchange as measured with the substrates [3-3H]crotonyl-CoA and unlabelled glutaconate and with glutaconate CoA-transferase as auxiliary enzyme. In the presence of free biotin or its methyl ester the soluble part catalysed the formation of crotonyl-CoA from glutaconyl-CoA (apparent Km for biotin 40 mM, Vmax 1% of the native decarboxylation reaction). This apparent reactivation was most likely caused by the carboxylation of free biotin. Based on these and other observations the following functions may be assigned to the different polypeptide chains of glutaconyl-CoA decarboxylase: biotin carrier (alpha), carboxytransferase (beta) and carboxylase, the actual sodium pump (gamma).     

Effects of Glyphosate on Metabolism of Phenolic Compounds VIII. Comparison of the Effects of Aminooxyacetate and Glyphosate

Aminooxyacetate (AOA), an in vitro inhibitor of phenylalanineammonia-lyase (PAL) and of some transaminases, was tested forcomparison with glyphosate's [N-(phosphonomethyl)glycine] effectson plant growth, PAL activity, and accumulation of hydroxyphenoliccompounds of three-day-old, dark-grown soybean [Glycine max(L.) Merr.] seedlings. Root-fed AOA (50 µM) and glyphosate(0.5 mM) caused similar decreases in growth rate and in accumulationof hydroxyphenolics, anthocyanin and chlorophyll. Together,these compounds were neither antagonistic nor synergistic inaffecting these parameters. AOA caused decreases in extractablePAL activity while increasing aromatic amino acid pools—theopposite of glyphosate's effects. In the light, the effect ofglyphosate on PAL and aromatic amino acids predominated overthose of AOA when the chemicals were given together. The effectsof AOA and glyphosate on most free amino acid levels were similar.In those cases in which the effects differed, glyphosate's effectpredominated over that of AOA when the chemicals were giventogether. The similarities and interactions of AOA and glyphosatein their effects on free amino acid profiles indicate that glyphosatesignificantly interferes with non-aromatic as well as aromaticamino acid synthesis. (Received April 6, 1982; Accepted June 28, 1982)     

Identification of Cytokinins in Root Exudate of the Rice Plant

Cytokinins, cis-zeatin and cis- and (trans-ribosylzeatin, wereidentified in the root exudate of the rice plant (Oryza sativa,indica cultivar IR-24) after several chromatographic separationsand combined gas-liquid chromatography-selected ion monitoring(GC-SIM) analysis. The presence of trans-zeatin ribotide wassuggested by enzyme hydrolysis, subsequent chromatographic separationand GC-SIM. The comparatively high content of the ribotide inthe root exudate suggests the form of cytokinins to be transportedfrom roots to other parts in the rice plant. (Received July 22, 1982; Accepted November 25, 1982)     

The Enzymic Decarboxylation of {gamma}-methyleneglutamic Acid by Plant Extracts

-Methyleneglutamic acid, an acidic amino-acid isolated fromgroundnut plants, was decarboxylated by enzymes present in extractsof Capsicum fruits, barley roots, and tulip leaves, and alsoby intact cells of Clostridium welchii S.R. I2. The amino-acidwas attacked in a similar manner to, but in all cases at a slowerrate than, l-glutamic acid. The nature of the enzyme responsiblefor the decarboxylation of -methyleneglutamic acid was furtherinvestigated using preparations from barley roots (which donot contain the amino-acid) and from tulip leaves (in whichthe amino-acid is normally present, together with larger amountsof its amide form, -methyleneglutamine). The effects of pH,inhibitors, and partial heat denaturation upon the enzyme systemspresent in the barley and tulip extracts indicated that a singleenzyme was responsible for the decarboxylation of both l-glutamicacid and -methyleneglutamic acid. Although the Cl. welchii rapidlydeamidated and then decarboxylated l-glutamine, -methyleneglutaminewas not attacked by the organism.     

A Manganese Protein from Thylakoids of a Cyanobacterium, Plectonema boryanum; Inhibition of Oxygen Evolution with Its Antibody

A manganese protein was solubilized from the thylakoids of thecyanobacterium Plectonema boryanum with a cholate-deoxycholatemixture, and purified to homogeneity by gel-filtration. Isolatedmanganese protein had a molecular weight of 13,000 and showedcatalase activity, which was insensitive to 3-aminotriazole.The antibody raised against the manganese protein inhibitedthe oxygen evolution using dichlorophenol indophenol (DCIP)as the electron acceptor by P. boryanum thylakoids, but notthe diphenylcarbazide-supported photoreduction of DCIP and ascorbate-supportedphotoreduction of methyl viologen in the presence of DCMU. Theseobservations suggest that isolated manganese protein is a componentof the oxygen evolving enzyme, water dehydrogenase. ¹ Dedicated to the late Dr. Joji Ashida, the first presidentof the Japanese Society of Plant Physiologists. (Received August 7, 1982; Accepted December 10, 1982)     

Distribution of Sorbitol, Neutral Sugars, Free Amino Acids, Malic Acid and Some Hydrolytic Enzymes in Vacuoles of Apple Cotyledons

Vacuoles of apple cotyledons (Malus pumila Mill. var. domesticaSchneid.) were obtained by purification with Ficoll densitygradient centrifugation after the protoplasts were lysed byboth osmotic shock and the addition of EDTA. High levels ofacid protease and carboxypeptidase activity were detected inthe vacuoles along with acid phosphatase, phosphodiesteraseand ATPase. The distribution of sorbitol and other sugars inthe vacuoles, the protoplast and extracellular free space wasdetermined. About 45, 60 and 90% of the sorbitol, glucose andsucrose, respectively, contained in whole tissue were foundin the extracellular free space, and 54% of the sorbitol inthe protoplast was detected in the vacuole. The sorbitol inthe vacuoles and the extravacuole of the protoplast was about80 and 70%, respectively, of the total sugar content of thecell. Arginine was the most abundant free amino acid in theprotoplast, and about 90% of it was contained in the vacuole.More than 80% of the total amino acids and 50% of the proteinwere also located in the vacuole, as well as most of the malate.The amounts of the total sugars, total amino acids, proteinand malate in the vacuoles were to 250, 400, 48 and 9 µg/10⁶cells, respectively. The results suggest that the vacuoles ofapple cotyledons contain a large pool of amino acids and proteinsrather than sugars, and have a close connection with proteinbody degradation. ¹ This paper is contribution A-l37 of the Fruit Tree ResearchStation. (Received January 20, 1982; Accepted May 18, 1982)     

Ethylene Production, 1-Aminocyclopropane-l-Carboxylic Acid Content and Its Conversion to Ethylene in Axial Segments of Dormant and Nondormant Cocklebur Seeds

During the imbibition of water, the change in the ethylene productionof axial segments of nondormant (ND) cocklebur (Xanthium pennsylvanicumWallr.) seeds paralleled the change in the content of free 1-aminocyclopropane-l-carboxylicacid (ACC), but not the change in conjugated hydrolysable ACCin the axes. Aminoethoxyvinylglycine, anoxia and a-aminoisobutyricacid inhibited ethylene production, the lattertwo compoundscausing the accumulation of free ACC. Administration of ACCgreatly enhanced ethylene production in the axes. Thus, freeACC seems to be a direct precursor of ethylene production inthe axial tissue of cocklebur seeds. Imbibed dormant (D) axes characterized by inferior ethyleneproduction had less ability to convert exogenously applied ACCto ethylene as compared to ND axes. But, there was little differencebetween the D and ND axes in the endogenous contents of freeand conjugated ACC. This suggests that the inferior ethyleneproduction found in detached D axes is associated with the lowactivity of an ACC-ethylene converting system. (Received December 17, 1982; Accepted April 30, 1983)     

Transfer RNA, a Possible Supplier of Free Cytokinins, Ribosyl-cis-zeatin and Ribosyl-2-methylthiozeatin: Quantitative Comparison between Free and Transfer Cytokinins in Various Tissues of the Hop Plant

Cytokinins in both free pool and tRNAs have been identifiedand quantified in the unfertilized cone, fertilized cone, seed,cotyledon, epicotyl and root of the hop plant (Humulus lupulusL. cv. Shinshu-wase) on the basis of combined gas chromatography-massspectrometry and combined gas chromatography-selected ion currentmonitoring analysis. Based on the quantitative comparison betweenfree pool cytokinins and tRNA cytokinins, it is suggested thatribosyl-cis-zeatin and ribosyl-2-methylthiozeatin might be derivedfrom tRNA catabolism and that the level of ribosyl-trans-zeatinmight be controled independently of the tRNA catabolism. ¹ Present address: Taisho Pharmaceutical Co. Ltd., Yoshini-cho,Omiya-shi, Saitama 330, Japan. (Received December 1, 1981; Accepted February 9, 1982)     

Changes in the Free Amino Compounds in Young Tomato Plants in Light and Darkness with particular Reference to {gamma}-Aminobutyric Acid

Tomato plants were grown to the five-leaf stage under uniformconditions in a growth room with a daily light period of 15h. Plants were sampled at intervals through 24 h periods andthe free ninhydrin-positive compounds determined in roots, bleedingsap, stems and shoots (mainly leaves), using ion-exchange columnchromatography and a lithium-buffer separation system. The compoundspresent and their range of concentrations are given for twooccasions: after illumination for 8 hand after 5 h of darkness. Data for -aminobutyric acid (GAB), glutamic acid, glutamine,alanine, aspartic acid and ammonia are summarized graphicallyfor all occasions and for all parts of the plant; asparaginefor sap only. The data were examined for correlations betweenthese substances for both light and dark conditions. Relative amounts of free acids were: root glutamine> glutamicand GAB > aspartic > alanine; bleeding sap glutamine >asparagine > GAB > aspartic> alanine; stem glutamine> glutamic > GAB and aspartic > alanine; shoot (leaf)GAB and glutamine > aspartic > alanine and glutamic. Patternsof change were as follows: in the root GAB and glutamic weresimilar and unlike glutamine; alanine did not change;sap ammonia,GAB and alanine were parallel, glutamine was similar to theseonly in light; in the stem glutamine and glutamic tended toaccumulate in parallel in light, but GAB did not; in the shoot(leaf) GAB and glutamine were similar except that the formeraccumulated more rapidly in the initial light period; glutamicacid and alanine were similar to each other but distinct fromGAB and glutamine. The relatively large amounts of GAB in tomato plants and themagnitude of the changes occurring in light and darkness seemindicative of its importance as a temporary storage productfor protein amino acids, but the factors controlling accumulationand utilization in different parts of the plant are unknown.     

Separation of Protochlorophylls Esterified with Different Alcohols from Inner Seed Coats of Three Cucurbitaceae

Reversed-phase high-performance liquid chromatography was usedto separate protochlorophyllide esters isolated from inner seedcoats of three Cucurbitaceae. At least 17 protochlorophyllsesterified with different alcohols were separated on an octadecylsilica (ODS) column from purified pigments of pumpkin (Cucurbitamoschata) and balsam pear (Momordica charantica) seeds witha single elution using 100% methanol within 22 min. The separationwas done according to the numbers of carbon atoms in the esterifyingalcohol of the protochlorophyll molecule with high resolutionand reproducibility. Gas chromatographic analysis of the pigment-esterifyingalcohols confirmed the presence of a number of protochlorophyllsesterified with different alcohols. This method permits rapid,efficient and quantitative detection and identification of chlorophyllsin complex mixtures. (Received May 17, 1982; Accepted September 6, 1982)     

Localization of Sorbitol Oxidase in Vacuoles and Other Subcellular Organelles in Apple Cotyledons

To investigate the function and subcellular localization ofsorbitol oxidase, free cells, protoplasts and isolated vacuolesof apple cotyledons (Malus pumila Mill. var. domestica Schneid.)were examined by differential and sucrose density gradient centrifugation.Twenty percent of the activity of sorbitol oxidase in the wholetissue was contained in the subcellular fraction (d=1.06) whichcorresponded closely to the main peaks of activity and proteinafter the recentrifugation of the 150,000?g pellet of rupturedvacuoles with a linear sucrose density gradient. The enzymethus appears to be derived from the tonoplast membrane. Thistonoplast membrane-bound sorbitol oxidase may play an importantrole in the transport of vacuolar sorbitol into the cytoplasm,rather than in the transport of sorbitol into the vacuole. About10% of the enzyme activity also occurred in the subcellularfraction having a density of 1.12–1.16, which coincidedwith the peaks of acid phosphatase and ATPase activities. Thereforesorbitol oxidase may also be associated with the plasma membrane.Furthermore, 30–40% of its activity was located in theinterspace between the cell wall and the plasma membrane, orperhaps attached weakly to them. These results suggest thatsorbitol is transported into the cytoplasm by being convertedto glucose by sorbitol oxidase. ¹ This paper is contribution A-138 of the Fruit Tree ResearchStation. (Received January 20, 1982; Accepted May 18, 1982)     

Flowering and Endogenous Levels of Benzoic Acid in Lemna Species

The occurrence of benzoic acid, a flower-inducing factor inLemna species, in L. paucicostata strains 151, 381, 321 andL. gibba G3 was established by several purification steps andfinal use of gas liquid chromatography-selected ion monitoring.Quantitative analyses of benzoic acid were made in non-floweringand flowering Lemna to determine differences in levels. Theendogenous level of benzoic acid was shown to vary dependingon culture conditions, but no positive correlation could befound between the endogenous level of benzoic acid and floweringof Lemna. (Received October 6, 1982; Accepted December 23, 1982)     

Changes in the Contents of Free and Protein Amino Acids in Tobacco Seeds and Placentae during Maturation

Changes in the levels of protein and free amino acids in theseeds and placentae of Nicotiana tabacum were studied duringseed development. Seed maturation was completed 24 days afteranthesis. During maturation, protein rapidly accumulated inthe seeds between the 6th and 18th day, along with an appreciablecompositional change in the protein amino acids as the proportionsof glutamic acid and arginine increased. The amount of freeamino acids in the seeds gradually decreased throughout maturation.The major free amino acid on the 6th day after anthesis wasglutamine, which then drastically decreased between the 6thand 12th day with increases of glutamic acid, proline, arginineand alanine. The latter amino acids decreased thereafter untilthe 24th day. On the other hand, the amount and composition of the proteinsin the placentae did not change significantly throughout seedmaturation. In the early stage of development, the major freeamino acids in the placentae were glutamine, asparagine andglutamic acid, while in the later stage asparagine was mostabundant. (Received March 12, 1982; Accepted August 16, 1982)