Volume 47, Issue 1-2

The aromatic compounds of more than 400 specimens of 33 species of the lichen genus Umbilicaria have been investigated by HPLC. Additionally to gyrophoric, hiascic, lecanoric, ovoic and umbilicaric acids, atranorin, norstictic and stictic acids, the new depside crustinic acid has been found. Structure elucidation of crustinic acid was made by means of HPLC cochromatography, NMR , UV and mass spectrometry. Now the secondary product patterns of Umbilicaria species are discussed and their significance for systematic purposes is shown.

Eight species of the genus Helichrysum, all of them endemic to Madagascar, were analyzed for lipophilic phenolic constituents deposited on the surfaces of leaves and stems. A total of 35 compounds was isolated and identified by spectroscopic means. Chalcones (10) comprised the largest group of compounds encountered, followed by flavonols (8), flavanones (7), flavones (4), benzophenones (3) and phloroglucinol derivatives (3). Many of the flavonoids were characterized by unsubstituted B-rings and/or C- or O-bound prenyl or neryl substituents. The Helichrysum species analyzed in this study are very similar to African species of this genus with regard to their lipophilic phenolic constituents. The phenolic com pounds studied thus appear to be conservative chemical characters of Helichrysum that were retained more or less unchanged during the origin of endemic taxa in Madagascar.

The aerial part of Lactuca viminea L. was examined for the presence of phenolic compounds. Six different flavonoids were isolated and identified by UV spectra, TLC, HPLCDAD , 1 H NMR and 13 C NMR. They were identified as apigenin, luteolin, quercetin, luteolin-7-O-glucoside, quercetin-3-O-glucoside and luteolin-7-O-glucuronide-6″methyl ester. Three phenolic acids were also detected: caffeic, ferulic and chlorogenic acid.

The composition of iridals, new triterpenoids from sword-lilies, and their fatty acid esters in roots, rhizomes and leaves of I. germanica Linn., I. pseudacorus Linn, and two different varieties of I. pallida Lam. was determined. Quantitative analysis of their total content in the various parts of the plants in the course of the year showed specific seasonal changes in rhizomes and leaves. Based on these results the possible significance of these compounds for the plants is discussed.

When grown in an iron-deficient medium Pseudomonas aptata produces both a desferri-ferribactin and a pyoverdin. The identical sequence of the peptide chain confirms the hypothesis that desferri-ferribactins are the biogenetic precursors of pyoverdins.

Protoheme and small quantities of heme a were isolated and purified from the wild type and the developmental mutant, C-2 A′, of Scenedesmus obliquus. In fast growing synchronized WT cells and greening cells of the mutant protoheme could be labeled with [1- 14 C]glutamate and [2- 14 C]glycine. The labeling of heme a was ambiguous and of minor quantity. During rapid chlorophyll biosynthesis the turnover of protoheme was determined by a pulse-chase experiment to have a half-life of 2 h. At the current stage of investigations the question, whether [2- 14 C]glycine is incorporated into protoheme via the Shemin pathway or via photorespiration or refixation of 14 CO 2 during general metabolism remains open.

Resorcinolic lipids, amphiphilic compounds from cereal grains show strong effects upon the activity of membrane enzymes. The concentrations for 50% inhibition of erythrocyte membrane acetylcholinesterase were in the range of 18 -90 μᴍ and were dependent on the length of the aliphatic side chain of the homologue and on the modification of hydroxyl groups in the benzene ring. Sulfonation of OH groups resulted in a drastic decrease of the inhibitory potency. The effect of resorcinolic lipids on the activity of Ca 2+ (calmodulin)-ATPase was the opposite. Up to concentrations of 50 μᴍ alk(en)ylresorcinols stimulated the activity of this enzyme and only slight inhibition (approx. 30%) was observed above 100 μᴍ. The results suggest that the effect of resorcinolic lipids might depend on their ability to alter the bilayer properties. Most probably these compounds decrease the mobility of membrane phospholipid molecules.

Resorcinolic lipids, significant constituents of cereal grains are long chain (C 13 -C 27) derivatives of 1,3-dihydroxy-5-alk(en)ylbenzene. It was shown that due to their phenolic nature resorcinolic lipids are effective in protection against fatty acids peroxidation. The phospholipid bilayers appeared to be also protected in the presence of these compounds. The maximal inhibition of peroxidation was 50-90% with IE 50 values in the range of 0.8 5-3.6 mol% of resorcinolic lipids.

Photosystem I complexes of Eremosphaera viridis De Bary (Chlorophyceae, Chlorococcales) were isolated and partially characterized. In the isolated PS I complexes, peptides of 64-60, 26, 23, 20, 15, 11 and 8.5 kDa could be detected. When Eremosphaera was grown under regular conditions the pigment composition of the isolated PS I complexes was similar to that found in PS I complexes from other green algae. However, when Eremosphaera was grown under nitrogen deficient conditions, PS I complexes contained the secondary carotenoids canthaxanthin and traces of astaxanthin and echinenone in addition to β-carotene, violaxanthin and lutein. The results presented indicate that the secondary carotenoids are associated with the LHC I of PS I. To our knowledge this represents the first report about the association of secondary carotenoids with light harvesting pigment protein complexes of green algae.

The effects of reversible manganese extraction on NADP + photoreduction were studied with higher plant subchloroplast preparations of photosystem II (PS II). Under anaerobic conditions, when the reaction centers (RCs) of PS II are “closed” (i.e. in the state [P680 Pheo] QA), and in the presence of ferredoxin-ferredoxin-NADP + reductase, NADP + reduction is observed at a rate of 0.8 -1.1 nmol/mg × chlorophyll × h. After complete removal of manganese from PS II, the rate of NADP + reduction is reduced 40 - 50-fold. Upon the addition of Mn at a concentration of approx. 4 Mn atoms per reaction center, the NADP + reduction is restored up to 85 -90% of the initial value. When half of this amount of Mn is combined with about 40 times of the equivalent concentration of other divalent ions (Ca 2+ , Sr 2+ , Mg 2+ etc.) the reaction is also reactivated. Dinoseb (10 -6 m) an inhibitor of electron transfer in PS II prevents NADP + photoreduction. It is concluded that under conditions when the first quinone acceptor, QA, is in its reduced state (Q A - ) electrons are transferred from reduced pheophytin (Pheo·̅) to NADP + , indicating that PS II can reduce NADP + without the participation of PS I. On the basis of these and literature data, an alternate pathway for electron phototransfer in PS II reaction centers of higher plants is suggested. Some problems concerning the Z-scheme are discussed.

The effect of exposure to strong white light on photosynthetic electron transport reactions of PS I and PS II were investigated in spinach thylakoids in the absence or presence of oxygen. Irrespective of the conditions used for photoinactivation, the damage to PS II was always much more than to PS I. Photoinactivation was severe under anaerobic conditions compared to that in air for the same duration. This shows that the presence of oxygen is required for prevention of photoinactivation of thylakoids. The susceptibility of water-splitting complex in photoinactivation is indicated by our data from experiments with chloride-deficient chloroplast membranes wherein it was observed that the whole chain electron transport from DPC to MV was much less photoinhibited than that from water. The data from the photoinactivation experiments with the Tris-treated thylakoids indicate another photodam age site at or near reaction centre of PS II. DCMU-protected PS II and oxygen-evolving complex from photoinactivation. DCMU protection can also be interpreted in terms of the stability of the PS II complex when it is in S2 state.

Low concentrations of cyanide affect the flash-induced oxygen evolution pattern in thylakoid preparations of the filamentous cyanobacterium Oscillatoria chalybea. At a cyanide concentration of 2 x 10 -6 ᴍ the effect lies exclusively on the S 2 - and S 3 -state whereas the subsequent flash yields and the steady state are barely affected, showing that the reaction center is not touched by the reagent. The influence of the chemical is characterized by the fact that at basic pH (7.5 -8.5) the deactivation mode of S 2 and S 3 is changed, showing that the cyanide-ion is the reactant. Whereas in the control the deactivation of S 2 yields as usual S 1 and S -1 , the deactivation pattern in the presence of cyanide shows that S 2 deactivates - without giving S 1 - , to a more reduced state, probably S 0 . In the flash pattern the two signal amplitudes of the first two flashes are lowered to zero in the presence of 2 x 10 -6 ᴍ cyanide and become uptake signals at higher cyanide concentrations. It is seen that in the presence of cyanide S 2 and to a lesser extent S 3 in the Oscillatoria system apparently react with oxygen. In tobacco thylakoids no effect of cyanide on the flash pattern itself is observed, even at higher concentrations (e.g. 10 -4 ᴍ), although the signal amplitudes of the entire pattern are affected at such concentrations. Photosystem II of cyanobacteria is characterized by the absence of the two extrinsic 16 kDa and 23 kDa peptides. As shown by mass spectrometry inside-out vesicles from tobacco thylakoids, in which these two extrinsic peptides have been removed by NaCl-washing, exhibit simultaneously flash-induced O 2 -evolution and an O 2 -uptake which both require the presence of CL - and Ca 2+ ions. Both the evolution signal and the uptake signal are DCMU sensitive and are inhibited by KCN. An amperometrically measured flash pattern of inside-out tobacco vesicles shows, if the assay is supplemented with CL - ions, the usual flash pattern known from the literature. The experiments seem to indicate that even in the washed inside-out condition the S-state system of higher plants is much less sensitive to ambient oxygen than in the phylogenetically older system of the filamentous cyanobacterium Oscillatoria chalybea.

We have sequenced 5-aminolevulinate dehydratase (ALAD; EC 2.4.1.24) of a plant. A fulllength cDNA clone (1727 bp) encoding this enzyme has been identified by immunoscreening a lambda gt 11 cDNA library of spinach. ALAD is not a plant-specific enzyme; however, the plant enzyme differs from the well known ALAD enzymes of bacteria, yeast and animals in structural and biochemical properties and in that it is located in the plastid. Differences and homologies can be traced back to the molecular level. The mature ALAD subunit, whose N-terminus was determined by automatic Edman degradation, is a protein of 367 amino acid residues and has a M r of 40,132. This figure is in the range of molecular weights of non-plant ALADs. The active centre is highly conserved and the same is true for the ion-binding domain, except that 4 cysteines of the non-plant enzymes (binding Zn 2+ ) have disappeared and a total of 6 aspartic acids meets the demands of Mg 2+ -binding. However, there are more distinct differences. Apart from a transit sequence of 56 amino acids targeting the plastid, the N-terminal part of the mature plant enzyme differs considerably from non-plant ALAD enzymes. It is rich in prolines and hydroxylated amino acids. The apparent M r on SDS-PAGE is 45,000 or higher, but up to now posttranslational modifications have not been found.

Lipoxygenases were highly purified from soybean seed, wheat seed and cucumber cotyledons. Substrate specificities of these lipoxygenases were studied by using an entire series of (ω6Z ,ω9Z )-C 13~ C 24 -dienoic acids as synthetic substrate analogues. Soybean lipoxygenase-1 and cucumber lipoxygenase showed broad specificities for these substrates while wheat lipoxygenase showed narrow specificities. Position of dioxygenation to each substrate was analyzed by high performance liquid chromatography. With soybean lipoxygenase-1 elongation of the distance between the terminal carboxyl group and the site of hydrogen removal in a substrate decreased the positional specificity of dioxygenation, while, with cucumber lipoxygenase, shortening the distance decreased the specificity. It was suggested that cucumber lipoxygenase and soybean lipoxygenase-1 recognized the terminal carboxyl group of a substrate to arrange it only in one orientation at the reaction center. In case of wheat lipoxygenase, recognition of the carboxyl group was thought to have crucial and essential role to secure the activity.

We analyzed the developmental regulation and the activation by wounding of several stressrelated genes in various parsley organs. The genes encode phenylalanine ammonia-lyase (PAL) and 4-coumarate: CoA ligase (4 CL), two enzymes of general phenylpropanoid metabolism; a flavonoid specific enzyme, chalcone synthase (CHS); a furanocoumarin specific enzyme, bergaptol O-methyltransferase (BMT); and a pathogenesis-related protein (PR 1). All genes or gene families exhibited high levels of expression in roots and during certain stages of leaf development. PAL, 4 CL and CHS were preferentially expressed in young leaves, BMT and PR 1 in old leaves. An appreciable increase in CHS mRNA levels was observed in wounded leaves. By contrast, root wounding led to a decrease in the existing CHS m RNA levels. A biphasic response (a decrease followed by an increase) to wounding was seen for BMT and PR 1 m RNA s in roots and for BMT mRNA in attached leaves. Using gene-specific oligonucleotide probes to measure the expression rates of three of the four PAL genes and of the two 4 CL genes separately we observed a differential behavior of the individual family members under many of the conditions tested. While PAL-3 was preferentially activated in wounded leaves and 4CL-1 in wounded roots, PAL-2 and 4C L-2 were primarily responsible for the high constitutive expression levels in roots and flowering stems respectively. Despite the differential expression of their individual members, the PAL and 4 CL gene families displayed very similar changes in the overall patterns of expression, reflecting their closely related functions in phenylpropanoid metabolism.

ATP-sulfurylase from the cyanobacterium Synechococcus 6301 was regulated in vivo during growth in batch culture. The activity was highest at the third day after inoculation, declining afterwards to a level found in resting cells. During growth with air supplemented with 2% CO 2 this activity increased 3-fold compared to controls grown with normal air as CO 2 source. Addition of either nitrite or urea enhanced ATP-sulfurylase activity about 2-fold, whereas cysteine and especially methionine decreased ATP-sulfurylase activity to 5% of controls without treatment. The ATP-sulfurylase was purified by conventional techniques using DEAE-cellulose chromatography and further separation on blue sepharose achieving a 250-fold increase in the specific activity. An apparent K ᴍ of 5 μᴍ for APS and of 40 μᴍ for pyrophosphate was determined with the purified enzyme fraction.

It is possible to obtain pollen germination and pollen tube growth in vitro if boric acid is present. In this work the effect was studied using as a semiquantitative parameter the mean length (l̄) of Lilium longiflorum pollen tubes. Pollen tube growth was examinated in dependence on boric acid, ortho-silicic acid, nucleic bases, Ca 2+ and Zn 2+ in 10% sucrose solution. The maximum of l̄ is obtained for concentrations between 2-20 ppm boron. The simultaneous supply of silicon added as water glass leads to a synergistic stimulation effect on pollen tube growth and facilitates branching. The silicon action is preceded of a pollen tube growth inhibition period during 3 h. Adenine and guanosine are able to substitute partially boron as pollen germination and pollen tube growth stimulator. Concentrations of 100 ppm adenine leads to half the boron effect. The same stimulation effect is obtained by guanosine. Ca 2+ can partially substitute boron as well. The stimulation action of boron is significantly attenuated by Zn 2+ and by the herbicide Dicuran. These and preceding results from physiological studies indicate that boron and silicon should be essential trace elements for the regulation of molecular biological processes.

Young poplar cuttings (Populus nigra L. cv. Loenen and P. maximowiczii Henry x P. nigra L. cv. Rochester) were exposed for six weeks in open-top chambers to realistic concentrations of pollutant mixtures: 1) control; 2) SO 2 /NO x ; 3)O 3 / NO x and 4)SO 2 /O 3 /NO x . In this sequence of fumigation variants, the degree of influence of the various parameters of the nitrogen metabolism and of premature leaf drop increased very frequently compared to the control plants, P. nigra L. proving to be the more sensitive species. The elevated Kjeldahl nitrogen content of the fumigated leaves was accompanied by either an increase in free amino acids or in total protein or, in the case of particularly large rises (SO 2 /O 3 /NO x variants), by increases in both substance groups. Proteolytic processes as a cause of the elevated content of free amino acids could be excluded to a large extent. A diminished de novo synthesis of proteins obviously led to a shift in the amino acid/protein relationship. In the younger fumigated leaves, the total concentration of free amino acids exceeded the values of the older leaves. The elevated amino acid content of the fumigated leaves was produced to a high degree by the glycolate pathway and the Krebs cycle. The increased turnover of the carbon skeletons was connected with a drastic starch degradation, especially in the older leaves. The interaction of the amino acid and carbohydrate metabolisms is probably an important regulator in the promotion of rapid growth of young leaves in order to compensate premature leaf loss.

Mature fruits from apple trees contain residual amounts of radiolabel derived from soil-ap- plied [3,5- 14 C]amitrole. This report deals with characterization of the soluble part of this radioactivity. By GC/MS of suitable derivatives 3-(1,2,4-triazol-1-yl)-2-aminopropionic acid was identified as a new metabolite of amitrole. Significant parts of radiolabel were incorporated into genuine plant products, indicating liberation of 14 CO 2 from the applied substance followed by reassimilation. Possible pathways of metabolism of amitrole within the system soil/apple tree are discussed.

The metabolism of [3,5- 14 C]3,5-dibromo-4-hydroxybenzonitrile (bromoxynil) was studied in Hordeum vulgare and Stellaria media. In excised barley seedlings the time course of metabolism was followed over 48 h and the distribution of radioactivity to various metabolic fractions measured. The metabolites, separated by TLC and HPLC, were identified by chemical and spectrometric methods. Bromohydroquinone and 2,6-dibromohydroquinone were found, indicating partly debromination and replacement of the nitrile group by hydroxyl. The major parts of bromoxynil and of the metabolites were recovered in glucosidic conjugates. In Stellaria media the same metabolites as in Hordeum vulgare were determined. Additionally, metabolites of unknown structures without bromine were detected.

By means of GC, GC/MS and GC-coupled electroantennogram techniques, (3Z,6Z,9Z)- 3,6,9-heneicosatriene (1), (3Z,6Z)-m-9,10-epoxyheneicosadiene (2) and (3Z,6Z)-cis-9,10- epoxyeicosadiene (3) were structurally elucidated as the biologically active components of the pheromone complex of Arctia caja (Lepidoptera: Arctiidae).

The inhibition by ryanodine of caffeine induced calcium release from actively loaded heavy sarcoplasmic vesicles has been studied in order to analyse the relation between the occupancy of the vesicular calcium release channels by ryanodine and channel function. Ryanodine binding was monitored with [ 3 H]ryanodine under ionic conditions favouring the establishment of binding equilibrium. Binding follows 1 : 1 stoichiometry yielding dissociations constants between 7 - 12 nᴍ and 12-15 pmol ryanodine/mg vesicular protein as maximum number of ryanodine binding sites. When ryanodine labeling was monitored by measuring the decline of the amplitude of caffeine induced calcium release 50% inhibition occurred at a free ryanodine concentration of 1 nM. At this concentration less than 10% of the available ryanodine binding sites are occupied. Caffeine induced calcium release is completely abolished when 3 pmol ryanodine/mg have reacted. A corresponding divergence between ryanodine binding and its effect on caffeine induced calcium release was observed when the initial rate of ryanodine binding was measured either by labeling the vesicles with [ 3 H]ryanodine or by following the decline with time of caffeine induced calcium release. Caffein induced calcium release declines four times faster than the fraction of unoccupied ryanodine binding sites, k = 4.3 x 10 4 ᴍ -1 s -1 versus 1.2 x 10 4 ᴍ -1 s -1 . The observed interrelation between the occupation of ryanodine binding sites and its effect on caffeine induced calcium release indicates that the caffeine sensitive calcium channel functions as an assembly of at least 4 ryanodine binding sites whereby the occupation of one site suffices to abolish calcium release. The stoichiometric composition appears to be not fixed but might change according to the size of the fraction of ryanodine receptors exhibiting caffeine sensitivity. The reported data were evaluated according to the algorithm derived by H. Asai and M. F. Morales, J. Biol. Chem. 4, 830-838 (1965) for the activity of a macromolecule and the extent of an inhibiting reaction.

Membrane order of mouse embryo fibroblasts and their ionizing radiation and chemically transformed counterparts was investigated using EPR spectroscopy after labeling the membrane of the cells with the fatty acid spin label, 5-nitroxy stearic acid. The EPR spectra were recorded at temperatures ranging from 18 to 38 °C for both control and transformed cells. The distance between the outer hyperfine splitting (2 T′||), which is used as an indicator of membrane order, varies in these two cell types. Below 28 °C, 2T′II is higher in transformed fibroblasts than in normal cells, whereas above this temperature membrane order is the same. Lipid analysis as carried out by the measurement of the cholesterol/membrane proteins and sphingomyelin/lecithin ratios, showed no difference in the amounts of the main membrane rigidifiers. These findings suggest that cell transformation of mouse fibroblasts induced by radiation or chemicals may produce alterations in the cell membrane, as evidenced by variations in its order at low temperature. These measured differences are presumably not attributable to its fatty acids composition but to its glycoproteins content, since changes in membrane regidifiers were not observed between normal and transformed cells.

N,N-phtaloyl-ʟ-glumatic acid (NPLG), a hydrolysis product of thalidomide, and the surfactant Tween 20 were co-administred intraperitoneally to pregnant NMRI mice on days 9a and 8⅔ p.c. at doses where the hydrolysis product as well as the surfactant alone induce minimal embryotoxicity. The combined substances (350 mg/kg NPLG + 1.0 ml/kg Tween 20) caused a potentiative teratogenic and an additive embryolethal response. Enhancement of the amount of Tween 20 in the combinations to 2.5 ml/kg led to the same potentiative teratogenic and, in addition, potentiative embryolethal response. Distribution studies using 14 C-NPLG showed a strongly increased NPLG content in the maternal plasma under the influence of 2.5 ml/kg Tween 20 but not after co-administration with 1.0 ml/kg Tween 20. The malformations obtained were of the same type in all groups and mostly located in the vertebrae and ribs. These axial and paraxial defects were frequently more severe and included more affected elements after application of the combined than of the single substances. Extension of our experiments with the combined substances to treatment days 8½ and 9½ p.c. revealed that the first affected segment shifts in caudal direction depending on an increasing embryonal age at treatment. Limb malformations occurred at a low frequency; most of them were of the same type as described in cases of human thalidomide embryopathy.