Band 48, Heft 9-10

Rapid progress in gene technology has allowed, on the one hand, insight to be gained into the complex molecular mechanisms of plant/pathogen recognition and the natural defence strategies of host plants. On the other hand, this technology can also be used for the controlled and efficient generation of genetic variability and for the identification of desirable genotypes, far beyond the possibilities of classical breeding. The first successful attempts have been made to improve resistance against pathogenic viruses, bacteria, fungi and insects by engineering transgenic plants. The majority of these strategies were based on constitutively expressing single proteins that are either toxic to the pathogen/pest, or interfere with its reproductive cycle. More refined strategies, which are at the stage of testing, try to mimic and modify naturally-evolved defence reactions of plants and, thereby, will potentially confer a more durable resistance to a broad range of pathogens

The epicuticular wax layer of Humulus lupulus contains homologous series of hydrocarbons, wax esters, benzylacyl esters, aldehydes, primary alcohols, fatty acids and the triterpenoids β-amyrin, α-amyrin both free and esterified with long chain fatty acids and also friedelanone. At 54%, primary alcohols form the largest component. No single class of lipids forms a pre­dominant component (i.e. more than 80%) of the wax layer. Under scanning microscopical examination, both upper and lower surfaces of H. lupulus leaves appear covered by a continuous way layer which is devoid of any sculpturing or crystalloids. Epidermal cells are arranged in dense lamellate or undulate cuticular folds. Four different types of trichomes were identified on the surfaces of hop leaves and tendrils. The adaxial leaf surface bears numerous single, uni­ cellular, silicified, pointed hairs as well as many glands. On the abaxial leaf surface there are very long pointed hairs arising only from the veins. Hairs on the hop tendrils arise from the veins but are completely different in shape having two sharp points extending at right angles to the hair-base.

Comparative phytochemical investigations were carried out on sporopollenins of the follow­ ing plant species: Bryophyta -Reboulia hemisphaerica and Polytrichum strictum; Pteridophyta -Equisetum arvense, Selaginella selaginoides micro-and megaspores; Gymnospermae -Cycas circinalis; Angiospermae -Typha angustifolia and Tulipa cv. “Apeldoorn”. Although the range of the analyses included species from the Bryophyta to the Angiospermae, fundamen tal correspondences could be detected in the 13 C NMR and FTIR spectra concerning the occur­rence of aliphatics, aromatics, ether and carbonyl/carboxylic functions in varying degrees. While the amount of aliphatics strongly dominated in the molecular structure of all other sporopollenins investigated here, it was found in sporopollenin of Equisetum arvense in a much lower extent. Despite the different morphology of Selaginella selaginoides micro-and mega­ spores, their sporopollenin spectra were nearly identical

The alkaloid composition of leaves of Lupinus linearis Desr. (3 ecotypes) and L. gibertia­ nus Smith (6 ecotypes) from Argentina and Brazil were studied by capillary gas-liquid chromatography and GLC-mass spectrometry (EI-MS). Both species are closely related according to morphological criteria. This view is supported by the alkaloid profiles which are very similar and share a series of new and uncommon alkaloids. Main alkaloids are lupanine, 13-hydroxylupanine, esters of 13-hydroxylupanine (e.g. 13-angeloyloxylupanine, 13-tigloyloxylupanine, 13-benzoyloxylupanine, 13-cis/trans-cinnamoyloxylupanine). Minor alkaloids are: sparteine, 11,12 -dehydrosparteine, ammodendrine, tetrahydrorhom bifoline, angustifoline, α-isolupanine, 5,6-dehydrolupanine, 11,12-dehydrolupanine, N-formylangustifoline, 13-cis-cinnamoyloxymultiflorine. New minor alkaloids which have been tentatively identified by GLC-MS are 13-hydroxy-17-oxolupanine and corresponding esters (13-an-geloyloxy-17-oxolupanine, 13-tigloyloxy-17-oxolupanine, 13-benzoyloxy-17-oxolupanine, 13-cis-cinnamoyloxy-17-oxolupanine, and 13-trans-cinnamoyloxy-17-oxolupanine).

Total activity of FBPase of Chlorella kessleri grown either in blue or in red light o f equal fluence rates differs (26.8 and 61.0 nmol · min -1 · mg protein -1 ) in crude cell extracts prepared at pH 8.5. The difference largely depends on FBPase II, an enzyme species assumed to be located in the chloroplast. FBPase II is strongly influenced by reducing agents. Addition of DTT, however, does not compensate for the difference in activities. Although it leads to a higher increase (197%) in the activity of the enzyme from blue than in that of the enzyme from red light conditions, the final activities (79.7 and 133.1 nmol · min -1 · mg protein -1 ) still differ by a factor of 1.67. In contrast to alkaline conditions the activity is approx. equal when crude cell extracts are prepared at pH 6.0. It is 62.7 nmol · min -1 · mg protein -1 in blue light and 70.7 in red light. Under these conditions DTT enhances both activities equally (189 and 201 nmol · min -1 · mg protein -1 ). These results indicate that the observed difference in enzyme activity at pH 8.5 does not result from different enzyme quantities. At pH 8.5 the mole mass of FBPase II is 88 kD a, while it is 1349 kD a at pH 6.0 under both light conditions. The smaller mole mass represents the monomeric form of the enzyme. The previously assumed pH-dependent oligomerization/dissoziation [8 ], which is generally proven, is therefore not responsible for the different activities of the enzyme from cells grown in red or in blue light. However, the monomeric form seems to possess a higher stability when prepared from cells from red light conditions. The different stability and activity of FBPase II under alkaline conditions are discussed in context with an altered carbohydrate metabolism in Chlorella kessleri grown autotrophically in different wavelengths of light

Putative intermediates of cardenolide biosynthesis, namely progesterone, pregnenolone, 5β-pregnane-3,20-dione or 5β-pregnan-3β-ol-20-one, were administered to light- or darkgrown shoot cultures of Digitalis lanata. The unsaturated com pounds were reduced to their respective 5 a-pregnanes, 5β-pregnane-3,20-dione was reduced to 5β-pregnan-3α-ol-20-one and 5β-pregnan-3β-ol-20-one was isomerized to the respective 3α-pregnane. Suspension cultures of Digitalis lanata, on the other hand, accumulated both the 3α- and the 3β-isom er of 5β-pregnan-3-ol-20-one when incubated in the presence of 5β-pregnane- 3.20-dione. When 5β-pregnan-3α-ol-20-one was administered the cultured cells accumulated large amounts of the 3β-isomer together with small amounts of 5β-pregnane-3,20-dione, which may be regarded as an intermediate during the isomerization reaction. Cell-free, buffered extracts from light-grown shoots were shown to reduce 5β-pregnane- 3.20-dione almost exclusively to 5β-pregnan-3α-ol-20-one when 0.05 m MgCl 2 were present in the incubation mixture. Under these conditions the formation of 5β-pregnan-3β-ol-20-one was inhibited. The enzyme activity could be recovered from m em brane-free supernatants. Optimum enzyme activity occurred at pH 7.0 and 42 °C. The energy of activation was 56.2 kJ/mol and the enzyme reaction was found to be NADPH -dependent. SH reagents were essential for enzyme activity. The enzyme seems to be specific for 5β-pregnan-3-ones since neither 5 a-pregnane-3-ones nor Δ4/Δ5-pregnenes were reduced. The NADPH : 5β-pregnane 3α-hydroxysteroid-5β-oxidoreductase described here may play a role in the regulation of cardenolide biosynthesis by removing precursors, such as 5β-pregnane-3,20-dione, from the pathway

To examine the assumption that condensed tannins exhibit uniform activity against herbivores, tannins extracted from four plant species with different in vitro protein binding abilities were fed to larvae of Trichoplusia ni in artificial diets. Larvae were measured for survivorship, growth and food consumption at three tannin concentrations (0.001%, 0.01% and 0.05% w. wt.). Larval performance was negatively affected by the tannins tested in a dose-response fashion, giving the different plant-derived tannins different activity thresholds. We did not find any significant correlation between the biological effects of these tannins and their protein binding abilities. This study indicates that different species of tannins do not act uniformly against a tannin sensitive herbivore, and that these differences can not be attributed to their different in vitro protein binding abilities

Fermentation of a mixture of 3,17α,21-triacetoxy-3,5-pregna-diene-20-one and 17α ,21- diacetoxy-4-pregnene-3,20-dione with Flavobacterium dehydrogenans ATCC 13930 led to 17α-acetoxy-21-hydroxy-4-pregnene-3,20-dione as the main product and the novel 17 aacetoxy- 6β,21-dihydroxy-4-pregnene-3,20-dione and 17α-acetoxy- 6α ,21-dihydroxy-4-pregnene- 3,20-dione as the minor ones. 6 -Hydroxylation was not found so far with this strain. The finding is in good accordance with the model of 6 -hydroxylation of steroids proposed for fungi by Holland et al

Propolis and propolis balsam from Canada was analyzed by gas chromatography-mass spectrometry and high performance liquid chromatography and the results compared. The compounds identified indicated that the main plant source was Populus from Section Aigeiros

Glandular trichomes in form of long stretched tubes are present on the lower leaf side of Quercus robur as shown by scanning electron microscopy. The glands contain an essential oil, which was isolated by steam distillation together with volatile waxy components of the leaves in an amount of 0.025% of fresh leaves. The product of steam distillation was analyzed by GC-MS. Identification of com pounds is based on comparison of their mass spectral data with those of authentic samples in combination with retention indices and MS data using the SeKoMS (Search Kovats Indices and Mass Spectra) Library. Altogether 184 components of the product of steam distillation were separated, 155 of which could be identified, another 7 were tentatively assigned. Three groups of substances according to their chemical composition are found: hexenyl derivatives and some acetals (32%); terpenes including monoterpenes (4% ), sesquiterpenes and diterpenes (21%); and alkane derivatives (35%). The residual 8% consist of benzyl alcohol, com pounds which stem from the degradation of carotenes, and miscellaneous constituents

During rapid extraction of chlorophylls from maize leaves under reducing conditions with methanol containing NaBH 4 , chlorophyll a remained unchanged but chlorophyll b yielded [7-hydroxymethyl]-chlorophyll b. The 3-vinyl group of chlorophyll b was also reduced forming significant am ounts, up to 60%, of [3-ethyl]-[7-hydroxymethyl]-chlorophyll b. This was unexpected since this reduction of the 3-vinyl group does not occur when isolated chlorophyll b is treated in an identical manner with methanolic borohydride. The vinyl-group of chlorophyll a is not reduced during the same extraction conditions suggesting that the presence of a formyl or hydroxyethyl group at C-7 is necessary. The presence of 8-hydroxyquinoline and NaBH 4 in equimolar (16.5 m M) concentrations strongly inhibits the reduction of the 3-vinyl group of chlorophyll b in leaf extracts

In the epidermal layers of rye primary leaves two flavone glycosides and several hydroxycinnamoyl esters are localized, whereas the mesophyll contains two flavone glucuronides and two anthocyanins. The concentrations of all these potential UV-B protective phenylpropanoid compounds could be reduced by application of 2-aminoindan-2-phosphonic acid (AIP), an efficient inhibitor of phenylalanine ammonia-lyase (EC 4.1.3.5). Photosystem II in the primary leaves of seven-days-old plants, grown in the presence of 20 μm AIP up to an age of 80 h, was more severely affected by UV-B than in control plants with the normal concentration of phenylpropanoid compounds. Damage of photosystem II in vivo was estimated by measuring the chlorophyll a fluorescence (parameter FJFm) of PS II. The results indicate an essential role of phenylpropanoid com pounds as UV-B protectants in rye primary leaves

TLC bioautography using precoated glass thin-layer plates impregnated with benomyl or carbendazim (MBC), and Cladosporium herbarum as a test fungus was evaluated as a facil way to detect plant secondary metabolites antidoting against benzimidazole fungicides. In addition to emodin and a-tocopherol from Polygonum sachalinense, three phenolics, 3,5-dihydroxy- 4-methylstilbene and 5-methoxy-6,7-methylenedioxyflavone from P. lapathifolium, and 2,6-dim ethoxybenzoquinone from P. thunbergii were isolated and characterized as new benzimidazole antidotes. Emodin exhibited the antidoting activity not only against benomyl but also against carbendazim (M BC), thiabendazole (TBZ), thiophanate-methyl and nocodazole. Furthermore, emodin showed antidoting activity against MBC in the wild-type Neurospora crassa and against diethofencarb in the mutant of N. crassa resistant to benzimidazole fungicides but highly susceptible to diethofencarb

Treatment of leaves of spinach, corn, and peas with the herbicides paraquat, amitrole or acifluorfen leads to oxidative stress resulting in a light driven drastically increased production of ascorbic acid radical (m̱onoḏehydroa̱scorbic acid, MDAA) which could be demonstrated by in vivo EPR analysis. A discrimination of the MDAA formation between the action of elec­tron uncouplers and catalase inhibitors can be achieved by observation of the radical rise kinetics. Significant MDAA signal intensities are detected in the darkness likewise. These signals are probably due to the action of ascorbic acid oxidase activated by membrane destruction.

Cyanoacrylate Inhibitors, Photosystem II, Hydrophobicity The secondary quinone binding site of photosystem II is also the binding site for many dif­ ferent herbicides. The 2-cyanoacrylate inhibitors are a potent class of electron transfer inhibi­tors which bind at this site and are extremely sensitive to minor structural variation. In order to understand their mode of binding, we have studied the interaction between the inhibitors and receptor in the D1 protein binding region (residues Leu 210 to Val 280) in terms of non­ bonded intermolecular forces. The intermolecular energy was calculated by van der Waals and electrostatic interactions after energy minimization of the combined structures to reduce inter and intramolecular strain. We have identified specific amino acid residues within the binding protein which are instrumental in binding the herbicide and have shown that the spatial arrangement of the herbicide functional groups within the binding site rather than their lipo­philicity is the determining factor in binding efficiency.

The 2-cyanoacrylate inhibitors are a potent class of herbicides which block electron transfer in photosystem II. The spatial arrangement of different functional groups are an important factor in determining activity and a number of derivatives have been used as stereospecific probes of the secondary quinone binding site. More than one region of stereoselectivity in the binding site has been identified which influences the interaction with specific groups of the inhibitor. We have studied the interaction of various stereoisomers of the cyanoacrylates with the binding site in the D1 protein (residues Leu 210 to Val 280) by determining the nonbonded intermolecular energies between the modelled structures calculated by van der Waals and electrostatic interactions after energy minimization of the combined structures to reduce inter and intramolecular strain and have found that the results reflect the experimentally determined data

Red and blue light pulses of 5 min applied together with 45 μM KNO 3 stimulated the nitrate uptake and reduction and the assimilation of ammonium in darkness in the red alga Corallina elongata. Nitrate reductase and glutamine synthetase activities were increased in darkness after the application of both red and blue light pulses. Red light produced a dramatic increase in enzyme activities after the first hour in darkness but after 4 h the effect of blue light pulses was greater. The photostimulation of nitrogen metabolism was correlated with light-regulated accumulation of soluble proteins. Nitrogen incorporation, assimilation of ammonium, accu­mulation of total proteins and the increment in total intracellular nitrogen was greater in N-limited algae (C:N = 17.3) than in N-sufficient algae (C :N = 10.3) after the application of light pulses and nitrate. Because the stimulant effects of red and blue light on N-metabolism were partially reversed by far-red light, the possible involvement of the photoreversible red/ far-red photoreceptor, phytochrome, is proposed. In addition, the blue-light effect seems to be mediated by a specific B-light photoreceptor besides phytochrome due to the different time course of the response and the extent of the stimulation after blue compared to red light pulses.

The translocation of ATP from the inside to the outside of the mitochondrial membrane has been studied in a male sterile (2219 A) and a male fertile (2219 B) line of sorghum. The translocation of ATP was found to be substantially lower in case of mitochondria from the male sterile line. The affinity of adenine nucleotides to the translocator proteins of the mito­ chondrial membrane was found to be almost one half in case of 2219 A as compared to 2219 B when the K m for ATP-ADP was determined by two different assays. It is proposed that the inadequate supply of ATP in the cytosol resulting from its inefficient translocation may con­ tribute to the male sterility in this line.

One copy of the psbA. gene which codes for the photosystem II reaction center D-1 protein from the thermophilic cyanobacterium Synechococcus elongatus has been sequenced. It is feas­ible that a disulfide bridge between D-1 Cys 212 and D-2 Cys 2I2 is reponsible for the thermo­ stability of the photosystem II reaction center from Synechococcus elongatus.

Quantitative structure-activity relationships (QSAR) for a series of N(7)-substituted guanosines as substrates for calf spleen purine nucleoside phosphorylase (PNP) were developed, and compared with those for acid hydrolysis of these analogues. There is no correlation between the rates for enzymatic phosphorolysis and acid hydrolysis, indicating that for the enzymatic reaction labilization of the glycosidic bond is not the only, nor the predominant, effect of N(7)-substitution. Multiple regression analysis of the enzymatic process revealed that optimal substrate properties (minimal Michaelis constant) are associated with the Taft electronic constant equal zero and a substituent size, parametrized by the Taft steric constant, smaller than that for a methyl group. These results support the hypothesis of protonation of the N(7)-position of the base by the enzyme as a catalytic mechanism for calf spleen PNP. Attention is drawn to the postulated similar mechanism of action of other purine N-glycosidases, including plant antiviral proteins which function as RNAN-glycosidases, and possibly some DNA N-glycosidases which function as repair enzymes

Among 4 strains of the purple bacterium Rhodospirillum rubrum only one did not contain trimethylamine N-oxide/dimethylsulfoxide reductase and was unable to grow under anaerobic dark conditions in fructose media supplemented with trimethylamine N-oxide (TMAO) or dimethylsulfoxide (DMSO ). By growing the bacteria in media treated with charcoal or supplemented with tungstate (2 mM ), TM AO/DMSO reductase activity was lowered to about 10% of the value of the untreated control. The enzyme was purified from the periplasmic protein fraction of strain S1 and was shown to contain 2 subunits (mol. wt. of 40,000 and 36,000, respectively) in a 1:1 stoichiometry. Although the affinity of the reductase to DMSO (K m - 0.67 mM) was much higher than to TMAO (K m = 24 m M ) , the catalytic activity with the latter substrate was about 10 times as high (90-110 μmol/min X mg protein) as with DMSO

Mofebutazon, Dihydrofolate Reductase, Non-Steroidal Antiinflammatory Drugs (NSAIDs) Mofebutazon, in contrast to phenylbutazon, inhibits dihydrofolate reductase in a concentration-dependent manner. An apparent for mofebutazon and dihydrofolate reductase in the presence of NADPH as electron donor and dihydrofolate as electron acceptor of approx­imately 0.2 mM was calculated.

Dolomite, a mineral composed of magnesium and calcium carbonates, potentiates the antitumoral activity of bleomycin: While 40 days after inoculation, no mice survived the Ehrlich ascites tumor burden, 44% of them survived it after bleomycin treatment, and 63% after a simultaneous treatment of bleomycin and dolomite. The beneficial antitumor effect of dolomite is probably related to its high content (12.8%) of magnesium