Activity of the tetramer and octamer of glutamine synthetase isoforms during primary leaf ontogeny of sugar beet (Beta vulgaris L.)

In extracts from the primary leaf blade of sugar beet (Beta vulgaris L.) we separated a chloroplastic isoform (GS 2) of glutamine synthetase (GS, EC 6.3.1.2) and one or two (depending on leaf age) cytosolic isoforms (GS 1a and GS 1b). The latter were prominent in the early (GS 1a) and late stages of leaf ontogeny (GS 1a and GS 1b), whereas during leaf maturation GS 2 was the predominantly active GS isoform. The GS 1 isoforms were active exclusively in the octameric state although tetrameric GS 1 protein was detected immunologically. Their activity stayed at a relatively constant level during leaf ontogeny; an increase was observed only in the senescent leaf. The activity of GS 2, however, changed drastically during primary leaf ontogeny and was modulated by changes in the oligomeric state of the active enzyme. In the early and late stages of leaf ontogeny when GS 2 activity was low (lower than that of the GS 1 isoforms), GS 2 was active only in the octameric state. In the maturing leaf, when GS 2 activity had reached its maximum level (much higher than that of the GS 1 isoforms), 80 of total GS 2 activity was due the activity of the tetrameric form of the enzyme and 20 was due to octameric GS 2. Tetrameric GS 2 was a hetero-tetramer and thus not the unspecific dissociation product of homo-octameric GS 2. In addition, GS 2 activity was modulated by an activation/inactivation of the tetrameric GS 2 protein. Due to an activation of the GS 2 tetramer, the activity of tetrameric GS 2 increased during leaf maturation from zero level 23-fold compared with that of GS 1a and 18-fold compared with that of GS 1b. Possible activators of tetrameric GS 2 are thiol-reactive substances. During leaf senescence, GS 2 activity decreased to zero; this decrease was due to an inactivation of the tetrameric GS 2 protein probably caused by oxidation.Abbreviations FLL final lamina length - FPLC fast protein liquid chromatography - GS glutamine synthetase - GHA -glutamyl hydroxamate - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase Dr. Roger Wallsgrove's (Rothamsted Experimental Station, Harpenden, UK) generous gift of GS antiserum is greatly appreciated.     

Enzymes in cardenolide-accumulating shoot cultures of Digitalis purpurea L.

In contrast to undifferentiated cell suspension cultures of Digitalis lanata, photomixotrophic shoot cultures of Digitalis purpurea accumulate cardiac glycosides in substantial concentrations. They are used to investigate enzymes of the cardenolide pathway. All cardenolides are 5-configurated. The progesterone 5-reductase and the 3-hydroxysteroid-5-oxidoreductase are present in shoot cultures but not in undifferentiated cell cultures. These enzymes provide precursors for cardenolides, whereas the presence of the progesterone 5-reductase, also present in shoot cultures, is discussed with regard to its role in phytosterol biosynthesis and may be attributed to the general steroid pathway. The progesterone 5-reductase had an activity maximum during the early growth period seven days after onset of cultivation, whereas the corresponding progesterone 5-reductase activity was highest on day 11. The maximum cardenolide accumulation was after 24 days. The enzyme activities present in crude extracts from shoot cultures were characterized with regard to their requirements for NADPH and NADH, pH-optimum, temperature optimum, affinity to their substrates and their localization in the cell. The progesterone 5-reductase was purified 769-fold.Abbreviations DW dry weight - FW fresh weight - PVP polyvinylpyrrolidone     

The legumin gene family: a reconstructed Vicia faba legumin gene encoding a high-molecular-weight subunit is related to type B genes

Nucleotide sequence information from a partial genomic clone, a cDNA clone, a RACE clone and a PCR fragment was combined to reconstruct the first reported complete gene sequence encoding a large legumin subunit, designated LelB3. The length difference to the well-characterized major legumin subunits is caused by an extended glutamin/glutamic acid-rich region encoded by the C-terminal part of the chain. Amino acid sequence comparisons reveal that gene LelB3 is more closely related to B-type than to A-type legumin genes of Vicia faba. Gene LelB3 is a member of a small gene family as indicated by published (Pich and Schubert, Biol Zbl 112 (1993); 342–350) and limited own data.     

The biosynthesis of rosmarinic acid in suspension cultures of Coleus blumei

Suspension cultures of Coleus blumei accumulate very high amounts of rosmarinic acid, an ester of caffeic acid and 3,4-dihydroxyphenyllactate, in medium with elevated sucrose concentrations. Since the synthesis of this high level of rosmarinic acid occurs in only five days of the culture period, the activities of the enzymes involved in the biosynthesis are very high. Therefore all the enzymes necessary for the formation of rosmarinic acid from the precursors phenylalanine and tyrosine could be isolated from cell cultures of Coleus blumei: phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, hydroxycinnamoyl:CoA ligase, tyrosine aminotransferase, hydroxyphenylpyruvate reductase, rosmarinic acid synthase and two microsomal 3- and 3-hydroxylases. The main characteristics of these enzymes of the proposed biosynthetic pathway of rosmarinic acid will be described.Abbreviations DHPL 3,4-dihydroxyphenyllactate - DHPP 3,4-dihydroxyphenylpyruvate - pHPL 4-hydroxyphenyllactate - pHPP 4-hydroxyphenylpyruvate - RA rosmarinic acid     

Interactions among juveniles of two freshwater crayfish species and a predatory fish

Two freshwater crayfish species, Astacus astacus L. and Pacifastacus leniusculus Dana, co-occur in some Swedish lakes. Observational studies indicate that the introduced, North American species P. leniusculus may gradually replace the native A. astacus, but the mechanism behind the replacement is not known. This study examined the direct effects of interspecific competition between the crayfish, and indirect effects of competitive interactions and fish (European perch, Perca fluviatilis L.) predation. Three different experiments with young-of-the-year (YOY) crayfish were performed. P. leniusculus was strongly dominant over similar-sized A. astacus in interference competition for shelter in a laboratory experiment. However, in a 35-day experiment in outdoor pools, A. astacus growth and survival were about equally affected by interactions with conspecifics and P. leniusculus. In contrast, P. leniusculus was significantly more affected by intraspecific competition than by competition with A. astacus, suggesting asymmetric competition between the two species. The presence of perch in outdoor ponds with mixed-species groups of the two crayfish species resulted in considerably higher predation rates on A. astacus than on P. leniusculus. Both species showed strong antipredator responses to perch by increasing refuge use. I suggest that higher perch predation rates on A. astacus originate from P. leniusculus being the superior species in interspecific competition for shelter. Because of displacement from refuges, A. astacus individuals become more exposed to the predator. This indirect effect of interactions among the two cray-fish species and the predator may be important in the observed in situ replacement of A. astacus by P. leniusculus.     

Behaviour of cyclic bank voles under risk of mustelid predation: do females avoid copulations?

Mustelid odours have been shown to suppress breeding in captive bank voles (Clethrionomys glareolus) from cyclic populations (Ylönen 1989; Ylönen and Ronkainen 1994). The mechanism behind the suppression is unknown. Based on a series of behavioural trials and breeding experiments with pairs of bank voles in breeding condition, we suggest that the primary cause for breeding suppression is a change in female mating behaviour. Experimental female-male pairs (n=34) exposed to mustelid odour decreased their general activity compared to control pairs (n=34). When encountering males in behavioural trials, females exposed to stoat odour were more aggressive and actively avoided precopulatory behaviours of males. No copulations were observed in experimental pairs compared to five in control pairs during the behavioural trials. Males actively approached females in general but male behaviour did not change under exposure to mustelid odours. We suggest that females are more vulnerable to mustelid predators than males and therefore actively avoid copulations in the (indirect) presence of mustelids. As well as this behavioural response, internal abortive mechanisms (cf. Bruce 1959) could play a role in the observed breeding suppression.     

Isolation and biochemical characterization of fusicoccin-insensitive variant lines of Arabidopsis thaliana (L.) Heynh

Arabidopsis thaliana lines have been isolated that are insensitive to the fungal toxin fusicoccin (FC). Initial screening was done by selecting for plants that either grew well on high concentrations of FC or did not respond to FC by increases in H⁺-extrusion. All selected plants were tested, in several additional rounds of screening, for binding to microsomal proteins of a ³H-labeled radioligand of fusicoccin. A novel assay allowing for the direct selection of individual plants exhibiting reduced binding of FC was developed and used as screening procedure. Independent variant lines (43) with stably expressed, reduced binding of FC were isolated and subjected to a detailed characterization of their binding sites. The lines could be subdivided into several distinct classes with respect to these characteristics. In class-I lines, the data indicate a partial conversion of high-affinity binding sites to a low-affinity state. In class-II lines, the affinity of the binding site to FC is strongly reduced while the number of sites, as well as several other biochemical parameters, is completely unchanged, suggesting a specific alteration in the properties of the fusicoccin-binding protein. In class-III lines, the ligand-binding protein complex, while retaining its high affinity, is destabilized at supraoptimal concentrations of FC (such as those used for screening). In wild-type plants, only the high-affinity binding site was detected. Combined, these data prove that the high-affinity sites represent the plant's FC receptor.Abbreviations A_o binding site concentration - FC fusicoccin - FCBP fusicoccin-binding protein - FCol 9-nor-8-hydroxyfusicoccin - K_D dissociation constant of the FCBP-radioligand complex We are grateful to Iris Sandorf and Gudrun Henrichs for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft, Bonn, Germany and by Fonds der Chemischen Industrie (literature provision).     

Changes in the content and constituents of the cuticular wax of Picea abies (L.) Karst. in relation to needle ageing and tree decline in five European forest areas

Cuticular wax composition of healthy and and declining mature Norway spruce trees [Picea abies (L.) Karst.] was investigated in five European forest areas. The amount of extracted wax and the content of alkanes and secondary alcohols were analysed as a function of the factors sample area (five areas, detailed below), needle age (current year to 2 years) and decline class (Class O to Class 2). Using a GC-MS, alkanes from C₂₀ to C₃₁ and the following alcohols were quantified: 10-nonacosanol, 5,10-nonacosandiol, 4,10-nonacosandiol and the triterpenol 24-methylenecycloartanol. According to our results, the total wax content as well as the alkane and alcohol content of waxes show a large variation corresponding to sample area and needle age. Ageing caused a highly significant increase in alkane content and a highly significant decrease in total wax and alcohol content. The decline class significantly influenced only the content of the long chain alkane C₃₁ (increase), the secondary alcohol 10-nonacosanol (decrease), and the triterpenic alcohol (decrease). Total wax weight was not influenced by tree damage. Thus, according to our results, needle ageing and progressive tree damage are correlated to different changes in the examined parameters.