Band 46, Heft 1-2

Over the last decade an intensive research on the regulation of gene expression in viral and animal systems has led to the discovery of cis-acting regulatory sequences, the identification of sequence-specific DNA -binding proteins (trans-acting factors), the characterization of protein domains involved in DNA -protein recognition and binding as well as in protein -protein interactions, and the cloning and sequencing of genes encoding regulatory proteins. The tre­mendous progress in this field is now being complemented by advances in our understanding of how plant genes are regulated. A wealth of data has accumulated in the past few years witnessing basic similarities in the transcriptional regulation of various eukaryotic genes, but also specific features of plant genes. This article collects presently available data, focusses on DNA -protein interactions in plant genes, particularly in light-regulated and “constitutively expressed” genes, reports on the isolation of plant genes encoding regulatory proteins, an dismeant to induce further activities in plant gene research.

Column chromatography of the acetone extract of the lichen Cladina macaronesica (Sephadex LH-20, silica gel and silver nitrate-im pregnated silica gel) afforded eight triterpenes identified by chemical and spectral m eans. α-Amyrenone, lupenone, taraxerol, taraxerone and /so-arborinol acetate were isolated for the first time from lichens and (-)-usnic acid and five mononuclear phenolic compounds were also obtained, four for the first time as natural products. The possible transformation of perlatolic acid into these phenolic compounds is briefly outlined.

The secondary product patterns of eleven Lasallia-species, L. asiae-orientalis Asah., L. brigantium (Zsch.) Llano, L. capensis (Frey) Llano, L. hispanica (Frey) Sancho & Crespo, L. membranacea (Laur.) Llano, L. papulosa (Ach.) Llano , L. pensylvanica (Hoffm.) Llano, L. pertusa (Rass.) L lano, L. pustulata (L.) Merat, L. rossica Dombr. und L. sinensis Vej are investigated by hplc. In addition to gyrophoric acid, lecanoric acid, umbilicaric acid, 7-chloroemodin, valsarin and skyrin, which were described before, hiascic acid and ovoic acid were found by means of hplc-cochromatography , UV -and mass spectrometry and the reac­tion of hiascic acid with acetone. The secondary product pattern of Lasallia-species is useful for systematic purposes. A key of the species is provided. The distribution of secondary prod­ucts within the thalli is discussed.

Reboulia hemisphaerica has been cultivated on Gamborg B5 medium with various sugars. Differentiated cultures grew best on agar medium with the addition of 0.5% sucrose and a light regime of 18 h light/6h dark . Callus was induced either by a combination of phytohor­mones (2 mg/l α-naphthylacetic acid + 1 mg/l kinetin) or by 4% glucose. The differentiated cultures on agar produced sesquiterpenes in a tenfold increased amount compared to undiffer­ entiated suspension cultures. Cultures kept in da rk produced no sesquiterpenes, but 5-hydroxy-7,8,4′-trimethoxyflavone was present whereas apigenin-7,4′-dimethylether was the only flavonoid in cultures cultivated in the light.

Using enantioselective MDGC the enantiomeric distribution of alkan-(alken-)2-yl-acetates from bananas is achieved. After simple acetylation their corresponding alcohols 2-pentanol, 2-hexanol, 2-heptanol an d (Z)-4-hepten-2-ol respectively are also exactly stereodifferentiated by the same method. All compounds investigated -esters as well as alcohols -exhibit a remarkable excess of the (S)-enantiomers.

Epicuticular and in tracellular flavonoids of Cistus laurifolius grown with and without UV radiation in a phytotron as well as under natural garden conditions in the field were studied. The amount of intracellular flavonoid glycosides of leaves receiving UV -A radiation was two fold higher than that measured in the absence of UV -A radiation , whether grown in the phyto­tron or in the field. Exposure of previously protected leaves to UV -A radiation increased the intracellular flavonoid glycoside content to that of unprotected leaves. The qualitative com­position of intracellular flavonoid glycosides showed a reduced amount of quercetin-3-galactoside to the myricetin monosides when the leaves were grown without UV-A radiation in the field and in the phytotron . Epicuticular flavonoid aglycones were not influenced by UV radiation significantly.

After inhibition of phenylalanine ammonia lyase by L- α-aminooxy-β-phenylpropionic acid, the constitutively formed formononetin 7-O-glucoside-6″-O-malonate is metabolized with the isoflavone aglycone being used as an intermediate in the elicitor-induced formation of pterocarpan phytoalexins in chickpea cell suspension cultures. In elicited cultures not treated with the inhibitor phytoalexins are synthesized de novo from phenylalanine. Therefore, in chickpea cells the constitutive isoflavone conjugate metabolism and the elicitor-induced pterocarpan formation show metabolic linkage under specific physiological conditions.

The chickpea pathogenic fungus Ascochyta rabiei when growing in a pectin containing min­eral medium secretes an exo-polygalacturonase (PG) and a pectin methyl esterase into the cul­ture medium. The induction of the PG is controlled by catabolite repression. The PG was puri­fied to apparent homogeneity from the culture fluid by acetone precipitation and chromato­graphy on a Mono S and a Mono Q column. The molecular weight of the purified glycoprotein enzyme in SD S-PAGE was about 70 kDa, whereas the Mr by gelfiltration it was estimated to be 53 kDa. Polygalacturonic acid is the only substrate of the PG whereas pectin is not degraded. The PG releases only galacturonic acid from polygalacturonic acid and no oligogalacturonides which might elicitate chickpea plants and trigger plant defense response. The enzyme is not inhibited by pectin, the released proteins or phenolics.

A yeast glucan elicitor causes the accumulation of the pterocarpan phytoalexins medicarpin and maackiain in chickpea (Cicer arietinum) cell suspension cultures established from seeds. A cell culture line from a chickpea cultivar resistant against its main fungal pathogen Ascochyta rabiei accumulates large amounts (944 nm ol/g fr. wt.) whereas a cell culture line from a susceptible cultivar accumulates only low amounts (38 nm ol/g fr. wt.) of the phytoalexins. This is consistent with differential accumulation of pterocarpan phytoalexins in intact plants [1], The first reactions in the pterocarpan-specific branch of biosynthesis are hydroxylation of the isoflavone intermediate form ononetin in position 2′ or 3′, catalyzed by microsomal cytochrome P-450 monooxygenases. Upon elicitation form ononetin 2′-hydroxylase undergoes a strong transient induction in the cell suspension culture of the resistant cultivar, whereas in the cell culture from the susceptible cultivar it is only slightly induced. In both cell suspension cul­tures the induction of cinnamic acid 4-hydroxylase and of form ononetin 3′-hydroxylase does not show a clear correlation with phytoalexin accumulation. Experiments with different elici­tor concentrations confirm that formononetin 2′-hydroxylase is much more induced in cell cul­tures from the resistant cultivar than from the susceptible one. It is concluded that the massive difference in phytoalexin accumulation between cell suspension cultures from the resistant and susceptible cultivar is determined mainly by the differential induction of form ononetin 2′-hydroxylase activity.

EPR investigations of spruce needles under in vivo conditions are capable of the detection of several radicals attributed to the plant photosystem . Furthermore, at room temperature an absorption due to ascorbic acid radical and other paramagnetic oxidation products can be de­tected additionally in the dark-adapted needles as well as under illumination. A systematic in­vestigation of the ascorbic acid signal intensity in spruce needles grown in the vicinity of air quality control units indicates two different oxidative mechanisms, depending on the state of illumination. Ozone plays a key role but is not the hazardous agent monitored by the ascorbic radical immediately. However, its signal reflects the present state of the antioxidative system in the needles and provides an early and simple diagnosis of the oxidative interactions of air pol­lutants with the photosynthetic apparatus of big trees.

The addition of 7-chloro-4-nitrobenzofurazan to isolated and activated CF 1 , creates a completely changed binding stoichiometry and subunit-distribution of bound modifier in contrast to the binding pattern in not-activated CF 1 . The activation of CF 1 , by dithiothreitol and heat results in the accessibility of three additional tyrosines in β-subunits and one additional tyro­ sine in α-subunits. Binding of pyridoxalphosphate to lysine in the active state suppresses the accessibility of the additional tyrosines, suggesting that PLP inactivates the ATPase by induc­ing a conformational change. Furthermore, two NBD -molecules are bound to γ-subunits when CF 1 , is activated. These two molecules may be bound to sulfhydryl groups of cysteines which become accessible to NBD after activation.

The association of the lipophilic tertiary amine and local anesthetic dibucaine with osmotically shocked chloroplasts of Spinacia oleracea L. cv. Monatol was investigated. Dibucaine, known as an effective inhibitor of thylakoid membrane energization and ATP synthesis, ex­hibited three distinct binding classes with chloroplasts: partitioning in the lipid phase of the membranes, electrostatic screening of negative electrical charges on the thylakoid surface and light-induced association of an as yet unknown nature. Evidence is presented that the mecha­nism of inhibition of the transthylakoid pH gradient, ∆pH, by dibucaine is distinct from ‘classical’ amine-type uncoupling: The inhibitory effect of dibucaine on ∆pH was independent of the initial strength of ∆pH. Light-induced dibucaine binding was independent of the volume of the intrathylakoid space and of the strength of ∆pH as varied by medium pH. Judged from a comparison of the data on dibucaine binding and on inhibition of ∆pH and photophosphorylation, dibucaine bound via partitioning in the membrane lipid phase is responsible for the uncoupler-like effects of the local anesthetic. A mechanism for the inhibition of thylakoid energization by local anesthetic amines is discussed.

The effect of K-picrate-18-crown-6 (crown) on the photoelectron transport activity of beet spinach thylakoid membranes was investigated. Addition of micromolar concentration of crown to thylakoid preparation inhibited p-benzoquinone, chloride-indophenol, methyl viologen supported Hill activities maximally by 75 per cent in a concentration dependent manner. However, the photosystem I catalyzed reaction remained insensitive to crown suggesting that crown specifically inhibits photosystem II electron transport. Addition of exogenous electron donors like hydroxylamine or diphenylcarbazide failed to restore the crown induced inhibition of photosystem II electron transport and lowering of steady state chlorophyll a fluorescence yield. These observations suggest that crown also inhibits photosystem II catalyzed electron transport after the donation sites of these exogenous donors. Washing of the crown pre-treated thylakoids with isolation buffer, relieved the crown inhibited electron transport activity, indicating that this inhibition is reversible. Furthermore, in hydroxylamine washed thylakoids which are devoid of O 2 evolution capacity, the hydroxylamine induced increase in chlorophyll a fluorescence of variable yield was quenched by the addition of crown. These observations suggest that crown affects the oxygen evolution and inhibits at a site close to photosystem II reaction centres.

X-ray crystallographic data for the highly potent cyanoacrylate photosynthetic electron transport inhibitor, (Z)-ethoxyethyl 3-(4-chlorobenzylamino)-2-cyano-4-methylpent-2-enoate, are presented. This compound has a particularly high affinity for the photosystem II (PS II) herbicide receptor with a p I 50 value of 9.5 (in the Hill reaction under uncoupled condi­tions with a chlorophyll concentration of 0.1 μg/ml). Data regarding the structure of small li­gands, such as this potent cyanoacrylate, which bind to the site with high affinity may be used to provide the basis for modelling studies of PS II/herbicide complexes. The X-ray data presented confirm the Z-stereochemistry of active cyanoacrylates and demonstrate the pres­ence of a planar core stabilized by an intramolecular hydrogen bond between the ester car­bonyl oxygen and a benzylamino hydrogen atom. In order to assess the importance of the benzylamino -NH -group in this type of cyanoacrylate, analogues containing a methylene group in its place were synthesized and found to be 100-and 1000-fold less active as Hill inhibitors.

Purified reaction center-B875 pigment-protein complex isolated from Rc. gelatinosus (I. Agalidis, E. Rivas, and F. Reiss-Husson, Photosynth. Res. 23, 249 - 255 (1990)) was further characterized. In the chromatophores, the quinone content was shown to be 6 menaquinones 8 and 16 ubiquinones 8 per reaction center, indicating that the pool contained both quinone types. Besides the primary (MK 8 ) and secondary (UQ 8 ) electron acceptors of the reaction cen­ter, the complex contains residual quinones from the membrane pool (about 3 MK 8 and 5 UQ 8 ) probably associated with the phospholipids. Apparent particle weight of the complex including bound detergent was 520 ± 46 kDa. The secondary quinone Q B was partially removed from the RC by treatment with 2 -3 % octaethyleneglycol dodecyl ether and 3 -4 mᴍ orthophenanthroline. Reconstitution experi­ments showed that UQ 6 , UQ 9 and UQ 10 could replace Q B but that MK 8 and MK 9 could not. It was concluded that Q B site has a clear specificity towards ubiquinone binding.

The cytoplasmic pH value of guard cells of Valerianella locusta was determined from the distribution of the weak acid [ 14 C]ABA between the medium and the cell, which was shown to be 8.1 ± 0.1. Treatment of guard cells in media with low pH (pH 5.0) and different concentrations of sodium acetate (0 − 5 mol m −3 ) caused a considerable acidification of the cytoplasm (6.6 − 7.7). Using the compartmental analysis we determined also the fluxes of ABA at the plasma membrane of guard cells (Φ ω ) and at the tonoplast of guard cells (Φ ᴄᴠ ) under steady state conditions. The flux of ABA from the cytoplasm into the vacuoles (tonoplast flux) was dependent upon the cytoplasmic pH. The data give evidence that ABA − is transported across the tonoplast by a saturable transport component. The ABA flux at the plasma membrane seemed to be independent of the cytoplasmic pH.

Propolis was analyzed by gas chromatography-mass spectrometry for both its headspace volatiles and for the less volatile components of its alcoholic extract (propolis balsam). 181 peaks were located of which 171 representing 150 compounds were identified, including 28 identified in propolis for the first time. The majority of compounds were typical of poplar bud exudate.

The surface lipids of the European mole consist of squalene, monoester waxes, triglycerides and cholesterol. As shown by GC/MS analysis, the fatty acid moieties of both monoester waxes and triglycerides belong to saturated unbranched , (ω-1)-and (ω-2)-methyl-substituted and unsaturated homologous series. Double bonds are located at the 7/8-and 9/10-position. Apart from the cis-also trans-isomers were found. The alcohol moieties of the monoester waxes consisted of saturated unbranched , (ω-1)-an d (ω-2)-methyl-substituted homologous series exclusively.

Changes in haemolymph biogenic amine levels of the cabbage armyworm , Mamestra brassicae, last instar larvae following injection of octopaminergic insecticides were investigated by using HPLC-ECD. When octopaminergic insecticides, chlordimeform or 2-(4-chloroo- toluidino)-2-oxazoline (AC-6), are injected, 4-tyrosine (TYR-4) is increased 10 min after injection. Clonidine injection also caused an increase of TYR-4. Based on these results, chemically induced insect stress is discussed.

This study deals with the structural perturbations that the antibiotic chlortetracycline hy­drochloride (CTC) can produce on phospholipid bilayers. Two multibilayer systems, one built-up of dimyristoylphosphatidylethanolamine (DMPE) and the other of dimyristoylphos-phatidylcholine (DMPC) were allowed to interact with different concentrations of CTC in the absence and presence of water. The study was carried out by X-ray diffraction methods and all experiments were done below the phospholipid main transition temperatures. The results showed that CTC produced significant perturbations on DMPC even in the absence of water, whereas they were much milder in DMPE .

Under conditions where cytochalasin B induces ATPase activity of monomeric actin (0.3 mᴍ MgCl 2 , 1 mᴍ EGTA , 30 (μᴍ cytochalasin B, 1 mᴍ ATP) the rate constant of the ex­change of actin-bound ε-ATP for free ATP is about 4 -6 times faster than steady state ATPase activity. When a stoichiometric ATP -actin complex is extracted with PCA (single turnover ex­periment) the apparent rate constant of P i generation is not faster than steady state ATPase activity. -The experiments suggest that the hydrolysis of actin-bound ATP and not the subse­quent release of hydrolysis products is rate-limiting during cytochalasin-induced ATPase activi­ty of actin.

Two arylphorin gene clones were inserted into the transposable p-element vector Carnegie 20 and injected into Drosophila rosy − mutants. Transponed animals were selected by their red eye colour and remated. We demonstrate by hybridization experiments the stable integration of the injected genes in the germ line of Drosophila. However, the incorporated genes are not expressed.

The presence of endogenous phosphorylase kinase and phosphorylase phosphatase in crude extracts of fat bodies from the cockroaches Nauphoeta cinerea and Periplaneta americana is demonstrated in vitro by activation/inactivation of glycogen phosphorylase under appropriate conditions. Fractionation of fat body extracts of both cockroach species on an anion-exchange medium results in the elution of three peaks with phosphorylase activity. According to their AMP dependency these activity peaks are designated as phosphorylase b (inactive without AMP), phosphorylase ab (active without AMP, but several stimulated with AMP) and phosphorylase a (active without AMP). It is shown chromatographically that incubating crude extracts of fat bodies from both cockroaches, under conditions where the phosphorylase kinase is active, results in all phosphorylase b being converted to the ab- or a-form , whereas under conditions where the phosphorylase phosphatase is active all phophorylase a is converted to the ab- or b-form . Endogenous phosphorylase kinase of N. cinerea crude fat body extract can convert vertebrate phosphorylase b into the a-form , and, conversely, vertebrate muscle p hosphorylase kinase and phosphorylase phosphatase, respectively, are able to convert partially purified N. cinerea phosphorylase aborb and the ab- und a-form , respectively. In resting cockroaches most of the phosphorylase activity resides in the b-form and only a small fraction (10% ) in the a-form , whereas between 26% (N . cinerea) and 35% (P. americana) occurs in the ab-form . Injection of endogenous hypertrehalosaemic peptides into N. cinerea (the decapeptide Bld-HrTH ) or P. americana (the two octapeptides Pea-CAH -I and II) causes interconversion of phosphorylase; after injection, mainly (60% ) phosphorylase a is present, while 25% and 15% exists in the ab- und b-form , respectively. Purification of the three phosphorylase forms from N. cinerea is achieved by anion-exchange chromatography on DEAE-Sephacel followed by affinity chromatography on AMP-Sepharose. The final specific activities are 2.1, 6.9 and 27.2 U /mg protein for the a-, ab- und b-form . The molecular mass of the active molecules on gel filtration is between 173,000 and 177,000, and SDS gel electrophoresis reveals a subunit mass of 87,100, suggesting a homodimeric structure for all three form s. Kinetic studies show hyperbolic saturation curves for the substrates glycogen and P i respectively, with Kᴍ-values of 0.021, 0.019 and 0.073% for glycogen and 8.3, 6.3 and 17.9 mᴍ for P i (a-, ab- and b-form ). Phosphorylase a exhibits a more or less hyperbolic response to AMP and needs 70 |iM A M P for m axim al stim ulation. The kinetics for the ab- and b-form s are sigm oidal and maximal activities are displayed at about 3 mᴍ (half-maximum activation as calculated from Hill plots are 55 and 280 μᴍ for the ab- und b-form , respectively). Caffeine is a strong inhibitor of the b-form , but has only a slight inhibiting effect (10 -20 % ) on the ab- and a-form in the presence of AMP.

Haemocyanin synthesis in the midgut gland of the crayfish, Astacus leptodactylus, was stud­ied in vitro. The addition of exogenous copper into the culture medium reduces the synthesis of total proteins and of haemocyanin. ʟ-lactate, uric acid and dopamine, which modify the affini­ty of haemocyanin towards oxygen, do not influence the biosynthesis of the polypeptide.

Several dimethyl-1,2-dithiolanes are identified in the anal sac secretion of the weasel. We compare and contrast the results with previously published work.