Growth and Dormancy in Lunularia cruciata (L.) Dum: VII. THE ISOLATION AND BIOASSAY OF LUNULARIC ACID

The extraction, purification, and isolation of the growth inhibitorpreviously postulated are described. Methanol extraction andseparation into acid, neutral, and basic fractions was followedby paper chromatography of the acid and neutral fractions withdistilled water, re-extraction with methanol, and thin-layerchromatography, the peak of inhibition being located at Rf 0.7–0.8(isopropanol: ammonia: water, 100:5:5), or Rf 0.3–0.4(chloroform: ethyl acetate: acetic acid, 60:40:5) Lunularia gemmae, grown directly on the chromatographic stripwith added nutrient solution, served as the most appropriateand direct bioassay. Area measurements after 5–10 days'growth yielded significant differences. Other bioassays included:Marchantia polymorpha gemmae, lettuce hypocotyl growth, cress-seedgermination, oat coleoptile, and radish cotyledon disc tests.An active inhibitor, i.e. dihydrohydrangeic acid, now named‘lunularic acid’, was isolated in crystalline form.Lunularic acid was found to increase with long-day treatmentof Lunularia thalli, though present even in short-day. Its concentrationcould be altered rapidly when daylength conditions were changed.The growth inhibition was linearly related to concentrationover the range from 0.1 to 10 ppm, very high concentrationsbeing lethal. Abscisic acid, though inhibitory to Lunulariain low concentrations, was not detected in extracts, and couldeasily be separated from lunularic acid.     

N-Acetylglucosamine Transfer Reactions and Glycoprotein Biosynthesis in Castor Bean Endosperm

N-Acetyl-[³H]glucosamine supplied to intact 3 d old castor beanendosperm tissue was incorporated into TCA-insoluble productpresumed to be glycoprotein. After an incubation time of 2 hthe major paniculate location of this product within the cellwas the endoplasmic reticulum. Cell-free preparations containingparticulate enzymes transferred N-acetyl-[¹⁴C]glucosamine fromUDP-N-acetyl-[¹⁴C]glucosamine into a fraction soluble in chloroform/methanol(2: 1, by vol), a fraction soluble in chloroform/methanol/water(10: 10: 3, by vol.), and an insoluble residue. Mild acid hydrolysisreleased the saccharide moieties from the lipids. Paper chromatographicanalysis of the released saccharides established that the C/M-solubleproducts contained both N-acetyl-[¹⁴C]glucosamine and N, N'-diacetyl-[¹⁴C]chitobiose.In contrast, N-acetyl-[¹⁴C]glucosamine released from the C/M/W-solubleproduct was contained in an oligosaccharide, probably in associationwith unlabelled mannose residues. The stimulatory effect ofdolichol monophosphate and the inhibitory effect of tunicamycinon saccharide-lipid synthesis indicated that N-acetyl-glucosamineis transferred to a glycopolymer by the established reactionsof the dolichol monophosphate pathway. The enzymes catalysingthe constituent reactions of this pathway were exclusively locatedin the ER.     

Studies on germination of chrysanthemum pollen II. Occurrence of a germinationpromoting substance

The in vitro germination of chrysanthemum pollen is promotedby adding floral organs, excepting anther, to a basal mediumconsisting of sucrose and boric acid. Some other plant organs,such as young fruit of tomato and onion bulb, are also effective.In Chrysanthemum morifolium, the percentage of pollen germinationin the presence of such plant tissues is two to three timesas high as in the control (10 %). In Ch. leucanthemum, it ishigher than 50 %, in contrast to the control in which no germinationis noted. This promotion of germination may be due to a substance extractablefrom the tissues with water, ether or methanol. The promoting substance is not identical with several knowngrowth regulators or with the Ca ion. (Received November 21, 1967; )     

Localization of 4-Chloroindole-3-acetic Acid in Seeds of Pisum sativum and Its Absence from All Other Organs

4-Chloroindole-3-acetic acid (4-Cl-IAA) was shown by GC-MS analysisto be present in immature and mature seeds of Pisum sativum,but not in any other organs of this plant. Its content was maximalat one week after anthesis and decreased as the seeds matured.Only indole-3-acetic acid (IAA) was detected in the other organsof P. sativum, its content being particularly high in the flowersand young pods during anthesis and the early pod set. (Received January 18, 1988; Accepted April 6, 1988)     

Hydrogen Peroxide-Dependent Oxidation of Flavonoids and Hydroxycinnamic Acid Derivatives in Epidermal and Guard Cells of Tradescantia virginiana L.

Luteolin, kaempferol, quercetin, caffeic acid and ferulic acidwere identified in acid-hydrolyzed epidermal strips of Tradescantiavirginiana using HPLC and spectrophotometry. The amount of flavonoidswas much smaller than that of cinnamic acid derivatives. Morethan 80% of the flavonoids were found in methanol extracts ofepidermal strips. Caffeic acid was found in both methanol extractsand the residues in nearly equal amounts, while more than 80%of the ferulic acid was found in the residues after methanolextraction. These data suggest that most of the ferulic acidand part of the caffeic acid bind to macromolecules as estersin the cell wall and that flavonoids are localized mainly inthe cytoplasm. The localization of esters of hydroxycinnamicacids in cell walls was ascertained by fluorometric analysis.These phenolic compounds were oxidized by H₂O₂ (0.025–1mM) in epidermal and guard cells and the oxidation was inhibitedby KCN and NaN₃: luteolin glycosides were less sensitive toH₂O₂ than quercetin and kaempferol glycosides in flavonoids.Ferulic acid esters were more sensitive to H₂O₂ than caffeicacid esters in hydroxycinnamic acid derivatives. On the basisof these data, the physiological significance of the oxidationof phenolic compounds by H₂O₂ is discussed. (Received October 9, 1987; Accepted February 3, 1988)     

Methanol Accumulation in Maturing Seeds

During in vitro growth and maturation of soybean seeds, cessationof embryo growth and dry weight accumulation occurred in thepresence of abundant C and N nutrients. Axis followed by cotyledontissues changed from green to yellow, and post-harvest germinationpotential declined if cultured after yellowing of axis tissues.A tissue specific accumulat;on of methanol occurred during thein vitro culture of immature seeds (i.e. initially 50 to 70mg fresh weight) to maturity in liquid medium. Methanol accumulatedto 3.0 g m^–3 or 50 µg seed^–1 in the medium,while methanol decreased from 37 to about 3.0 µg g^–1fresh weight in cotyledons. By contrast, axis tissues increased20-fold in methanol concentration to 90 µg g^–1 during20 d in culture. Ethanol was present only in trace amounts inaxis tissues and medium. Addition of exogenous methanol vapourto in situ grown seeds during precocious maturation decreasedsubsequent seedling vigour and germination with increasing levelsof exposure. Methanol accumulation in axis tissues during thegermination phase was not correlated with high temperature andtissue water content treatments which simulated pre-harvestdeterioration of seeds. However, the accumulation of methanolduring in vitro seed development and maturation in liquid culturemay contribute to reduced post-harvest germination performance. Key words: Soybean, Glycine max, seed maturation, in vitro, methanol     

Requirement of sodium chloride for the action of gibberellic acid in stimulating hypocotyl elongation of a halophyte, Salicornia herbacea L.

NaCl stimulated hypocotyl elongation of the halophyte Salicorniaherbacea L. grown either in light or dark. Its optimal concentrationwas around 0.1–0.2 M and its promoting effect was muchmore prominent in the dark. Gibberellic acid at 10^–5 Mstimulated hypocotyl elongation in light but not in the dark.Indole-3-acetic acid and kinetin were ineffective in promotinghypocotyl elongation. In light, gibberellic acid and NaCl synergisticallyenhanced hypocotyl elongation when both were given simultaneously.The action of NaCl could be replaced by KCl, but not by mannitol.Osmotic pressure of the epidermis of the Salicornia hypocotylincreased in response to gibberellic acid and/or NaCl treatment.Na⁺ content in the hypocotyl increased with NaCl application.Gibberellic acid and NaCl when given alone increased the extensibilityof the hypocotyl cell wall. Synergistic interaction in increasingthe extensibility was observed between gibberellic acid andNaCl. Stress-relaxation analysis of mechanical properties ofthe hypocotyl wall revealed that gibberellic acid and NaCl actedsynergistically in decreasing minimum relaxation time. Basedon these results, a possible mechanism by which gibberellicacid and NaCl regulate hypocotyl elongation of Salicornia herbaceaL., a typical halophilic plant, is discussed. ¹ Present address: Laboratory of Biology, Tezukayama College,Gakuen Minami, Nara 631, Japan. (Received June 13, 1978; )     

Existence of a Common Glycoprotein Homologous to S-Glycoproteins in Two Self-incompatible Homozygotes of Brassica campestris

S-Glycoproteins (S-locus-specific glycoproteins) in Brassicaspecies are present only in stigmas and thought to play an importantrole in self-incompatibility system. The stigma extract containsalso several other glycoproteins reacting with the antiserumto S-glycoproteins, among which some glycoproteins from S₈S₈-and S₉S₉-homozygotes have the same pI value. Both of the glycoproteinswhich were tentatively termed NS₈- and NS₈S₉-glycoproteins,respectively, were isolated and analyzed. Those were revealedto be identical. Its amino acid sequence was homologous withthe S-glycoproteins in Brassica species. The NS-glycoproteinswere expressed at the same time and only in stigma as S-glycoproteins. (Received July 19, 1988; Accepted September 7, 1988)     

Chlorophyll Metabolism in Higher Plants VI. Involvement of Peroxidase in Chlorophyll Degradation

The following phenolics were found to be essential for peroxidase-dependentchlorophyll bleaching: 2,4-dichlorophenol (DCP), p-coumaricacid (HCA), phenol, p-hydroxyphenylacetic acid, p-hydroxybenzoicacid, p-hydroxyacetophenone, resorcinol and umbelliferone. Mostof them are monophenols with electron-attracting groups at thep-position. The short-lived radicals generated by horseradishperoxidase (HRP)-phenolics-H₂O₂ reaction might be involved inthis reaction. Tobacco leaf enzyme preparation with peroxidaseactivity for guaiacol could also degrade chlorophyll with suchphenolics. In addition, tobacco leaf methanol extract couldsubstitute for chlorophyll bleaching as an electron donor inthe absence of phenolics. In place of free H₂O₂, the glycolate-glycolateoxidase (GOX) system could degrade chlorophyll in [peroxidase$phenolics]-dependentbleaching. This chlorophyll bleaching system was inhibited by peroxidaseinhibitors, radical scavengers, reducing reagents, and carotenoids.Ascorbate and glutathione stopped chlorophyll bleaching withGSSG reductase and NADPH. The role of ascorbate and glutathionein peroxidase activity for controlling the chlorophyll degradationrate is discussed. (Received January 28, 1985; Accepted July 23, 1985)     

Nitrobacter agilis Cytochrome c-550: Isolation, Physicochemical and Enzymatic Properties, and Primary Structure

Nitrobacter agilis cytochrome c-550 was purified to an electrophoreticallyhomogeneous state, and some of its properties were determined.The cytochrome showed an absorption peak at 410 nm in the oxidizedform, and peaks at 416, 521 and 550 nm in the reduced form.Its isoelectric point was 8.1 at 5?C. Analysis of the aminoacid composition showed that the cytochrome molecule was composedof 108 amino acid residues, 16 of which were lysine residues. The cytochrome reacted rapidly with N. agilis cytochrome c oxidaseand yeast cytochrome c peroxidase and more slowly with Pseudomonasaeruginosa nitrite reductase and bovine cytochrome c oxidase.The reactivities with these redox enzymes suggested that thecytochrome might be an evolutionary stage between bacterialand eukaryotic cytochromes c. The primary structure of the cytochrome from the N-terminusto the 85th residue was determined. The N-terminal sequencewas homologous to the corresponding portion of the primary structureof horse cytochrome c. ¹ Present adress: Department of Chemistry, Faculty of Science,Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo,152, Japan. (Received December 3, 1981; Accepted January 28, 1982)     

3-Deoxy-D-arabino heptulosonate 7-phosphate synthase from Zea mays: General properties and regulation by tryptophan

In extracts from Zea mays shoots, the presence of thiol compoundsin the extraction buffer was necessary to get an active 3 deoxy-D-arabinoheptulosonic acid 7-phosphate (DAHP) synthase. Its pH optimumfor activity was about 7.5. Of the different cations tested,only Mn⁺⁺ was an activator. Enzyme stability was optimal inTris-HCl buffer, pH 7.5, that contained a reducing agent, Mn⁺⁺and a polyol. Contrary to other reports, phosphoenolpyruvate(PEP) did not stabilize the preparation significantly. The synthaseexhibited high affinities for both erythrose-4-phosphate (Km:0.24 mM) and PEP (Km: 0.31 mM). Its specific activity was highestin young shoots. Corn DAHP synthase was inhibited in vitro by tryptophan. Moreover,the enzyme was retarded on a tryptophan agarose affinity column,but it was removed with the bulk of protein from the same supportwhen eluted with buffer containing tryptophan. Inhibition whichwas easily lost during storage at 4°C was pH dependent andincreased during development. Maximal inhibition, about 60%with 1 mM tryptophan, was observed in extracts from 8 day-oldshoots. Phenylalanine and tyrosine were not inhibitory, andno synergistic effects were observed when the aromatic aminoacids were tested in combination. Isoenzymes could not be demonstrated. (Received April 23, 1980; )     

Alcohol Stress on Soya Bean Seeds

When soya bean seeds were exposed to pure aliphatic alcohols,the shorter alcohols were the most damaging (methanol > ethanol> n-propanol > n-butanol); when the alcohols were appliedin equal volumes of water, the opposite was found (n-propanol> ethanol > methanol), leakage of solutes from the pre-treatedtissue during subsequent imbibition in water was associatedin each case with the loss of germination and a decline in axisgrowth. Damage by the pure alcohols was related to the extentof their penetration and the amount of phospholipid eluted,injury caused by alcohols in the presence of water did not exhibitthese functions. It is proposed that damage to seeds by alcoholsis due to the elution or displacement of cellular phospholipidsand possibly the partial denaturation of membrane proteins Membranedamage is considered to be a prime cause of injury to the seed. Glycine max (L.) Merr., soya bean, seed, denaturation of membranes, alcohols, phospholipids     

Carotenoid Pigments of some Lower Ascomycetes

Protomyces pachydermus and P. inouyei contains ß-,-carotene and lycopene, with ß-carotene predominating.Taphrina deformans and T. communis contains no carotenoid pigmentsalthough the colour of methanol extracts of all four speciesis yellow. A quick way of differentiating between these twogenera is by showing the presence of carotenoid pigments inProtomyces and absence of these in Taphrina. It is suggestedthat a knowledge of individual carotenoid pigments in fungimight be useful as a taxonomic tool among fairly closely relatedspecies.     

Trypanosoma rangeli sialidase: cloning, expression and similarity to T.cruzi trans-sialidase

Sialidases are hydrolytic enzymes present from virus to highereukaryotes, catalyzing the removal of sialic acid from glycoconjugates.Some protozoa Trypanosomatidae secrete high levels of sialidaseinto the medium. We have now purified the secreted sialidasefrom Trypanosoma rangeli Its N-terminal sequence reveals 100%identity with the corresponding region of the trans-sialidasefrom T.cruzi Trans-sialidase, although homologous to viral andbacterial sialidases, displays a novel sialyltransferase activityand is involved in host cell invasion. Several homologous trans-sialidase-likegenes were cloned from genomic DNA of T.rangeli, and groupedin three subfamilies. Active siali-dase-encoding genes werefound in one of them. The re-combinant sialidase shows similarproperties to those of the native enzyme, including undetectabletrans-sialidase activity. Nevertheless, it has an overall identityof 68.9% with the catalytic domain of T.cruzi trans-sialidase,increasing to 86.7% admitting conservative substitutions. Onlythree other eukaryotic sialidases have been previously cloned,none of them showing significant homology to trans-sialidase.The isolation of a highly similar sialidase is relevant to furtheridentify the molecular determinants allowing trans-sialidaseactivity. As a first approach, chimeric constructs between sialidaseand trans-sialidase were generated, one of them rendering asialidase with three times lower K_m than the natural enzyme. eukaryotic sialidase gene family glycosidase parasite sialic acid     

Enzymatic Conversion of Pheophorbide a to the Precursor of Pyropheophorbide a in Leaves of Chenopodium album

Soluble proteins extracted from leaves of Chenopodium albumcatalyzed the conversion of pheophorbide a to a precursor ofpyropheophorbide a, putatively identified as C-13²-carboxyl-pyropheophorbidea. The precursor was then decarboxylated non-enzymatically toyield pyropheophorbide a. Soluble proteins and pheophorbidea, as the substrate, were required for the formation of theprecursor, and boiled proteins were enzymatically inactive.The maximum rate of conversion of pheophorbide a to the precursoroccurred at pH 7.5. The K_m for pheophorbide a was 12.5 µMat pH 7.0. Both pheophorbide b and bacteriopheophorbide a couldserve as substrates, but protopheophorbide a could not. Formationof methanol was detected during the enzymatic reaction, an indicationthat the enzyme is an esterase. Among seven alcohol analogstested, only methanol inhibited the enzymatic activity uncompetitively,with a K₁ of 71.6 mM. Mass-spectrometric (MS) analysis of theprecursor yield a peak at m/z 579 that indicated the releaseof a methyl group from pheophorbide a. It appears thereforethat the enzyme catalyzes the demethylation of the carbomethoxygroup at C-13² of pheophorbide a by hydrolysis to yield methanoland the precursor, C-13²-carboxyl-pyropheophorbide a, whichis converted to pyropheophorbide a by spontaneous decarboxylation.We have tentatively designated the enzyme "pheophorbidase".The presence of the enzyme was dependent on plant species andit was expressed constitutively. ¹Present address: Faculty of Science, Shizuoka University, Ohya,Shizuoka, 422 Japan     

Germination Inhibitors in Fruit Extracts of Red Beet (Beta vulgaris cv. rubra

Germination inhibitors in methanol and water extracts of redbeet fruits (Beta vulgaris cv. rubra L.) have been studied usinglettuce and red beet seed germination as bioassays. The methanolextracts contained substances which inhibited lettuce seed germination,but had no effect on the germination of red beet seeds. Germinationof both lettuce seeds and of water-leached or sulphuric acid-treatedred beet seed balls were inhibited by the water extracts. Theconcentrations of ammonia, ferulic acid, and oxalate in thewater extracts were much lower than required for inhibitionof red beet seed germination. The water extracts contained,however, large amounts of inorganic ions, and the results clearlydemonstrated that the inhibitory effect of the water extractson red beet seed germination was mainly due to the content ofsuch inorganic ions.     

Purification and Characterization of a Cytochrome P450 (trans-Cinnamic Acid 4-Hydroxylase) from Etiolated Mung Bean Seedlings

A Cyt P450 (P450_C4H) possessing trans-cinnamate 4-hydroxylase(C4H) activity was purified to apparent homogeneity from microsomesof etiolated mung bean seedlings. Upon SDS-polyacrylamide gelelectrophoresis, the purified preparation gave a single proteinband with a molecular mass of 58-kDa. Its specific P450 contentwas 12.6 nmol (mg protein)^–1. Using NADPH as electrondonor, purified P450_C4H aerobically converted trans-cinnamicacid to p-coumaric acid with a specific activity of 68 nmolmin^–1 nmol^–1 P450 in a reconstituted system containingNADPH-Cyt P450 reductase purified from the seedlings or rabbitliver microsomes, dilauroyl phosphatidylcholine, and cholate.This specific activity is by far the highest for reconstitutedC4H systems so far reported and provides direct evidence thatC4H activity is actually associated with a P450 protein. Inthe oxidized state P450_C4H showed a typical low-spin type absorptionspectrum with a Soret peak at 419 nm. A partial spectral shiftto the high spin state was observed when trans-cinnamic acidwas added to oxidized P450_C4H. By spectral titration, the dissociationconstant of the cinnamic acid-P450_C4H complex was determinedto be 2.8 µM. This value is similar to the K_m value (1.8µM) for trans-cinnamic acid determined in the reconstitutedsystem. (Received November 20, 1992; Accepted February 17, 1993)     

Purification and Characterisation of the Light and Dark Forms of Phosphoenolpyruvate Carboxylase from the Dicot Plant Amaranthus viridis L. An Examination of Its Kinetic and Regulatory Properties in the Presence of Water-Alcohol Binary Solvents

The light and dark forms of phosphoenolpyruvate (PEP) carboxylase(PEPC) from the dicot plant Amaranthus viridis L. were purifiedand their kinetic properties were studied in water-based orbinary alcohol-water solvents. At pH 7.3, the specific activityof the purified light form was about 2.7-fold higher than thatpresented by the dark form of PEPC under optimal conditions,while K_m remained virtually unchanged in both forms. The enzyme'slight form was better activated by glucose 6-phosphate and lessinhibited by L-malate than the dark PEPC. From the organic solventsstudied, methanol showed the most important effect, enhancingPEPC activity by two-fold at 20% (v/v). Ethanol, ethylene glycol,tert-butanol and 2-propanol were also activators to a lesserdegree, but at high concentrations (typically greater than 20%,v/v) the effect was reduced or turned to inhibition. K_m (PEP)was reduced by an order of magnitude in the presence of 20%(v/v) methanol (i.e. from 0.32 to 0.022 mM for the light formof the enzyme). The inhibitory effect of malate at low PEP waslessened by methanol for both forms (i.e. I₅₀ 0.25 mM in aqueousmedium to 0.48 mM in binary mixture for the dark form), whileglucose-6-P activation of PEPC was not affected by methanol.The results suggest that the kinetics of PEPC in a medium thatmimics more closely in vivo conditions are different from thoseobserved by standard procedures consisting of aqueous media,and provide a new insight on the properties of PEPC as relatedto its regulation in vivo. (Received June 26, 1995; Accepted August 24, 1995)     

THE OCCURRENCE OF THE UPPER JURASSIC BIVALVE MALAYOMAORICA MALAYOMAORICA (KRUMBECK) ON THE ORVILLE COAST, ANTARCTICA

Malayomaorica malayomaorica is an important Upper Jurassic bivalvein the southern hemisphere: widely distributed in strata ofEarly - Middle Kimmeridgian age, it is a zone fossil of considerablepotential for regional biostratigraphic correlations. Its commonoccurrence in the Latady Formation of the Orville Coast, Antarctica,indicates that at least part of this stratigraphic unit hasa Kimmeridgian age. Although its precise taxonomic status remainsin some doubt, it would appear to be the earliest buchiid-likebivalve so far recorded from the southern hemisphere. Its verywide distribution around the margins of Gondwana is similarto that established for species of the Late Jurassic bivalvegenera Retroceramus, Buchia and Anopaea (Received 13 April 1981;