Purification and PCR-based cDNA cloning of a plastidial n-6 desaturase

A plastidial membrane-bound n-6 desaturase from spinach (Spinacia oleracea) was purified from chloroplast envelope membranes by anion exchange, cation exchange and ferredoxin-affinity chromatography. The molecular mass of the protein was estimated by SDS-PAGE to be 40 kDa. The highest specific activity of the desaturase in the final preparation was 196 nmol/min per mg protein with free oleic acid as the substrate. The N-terminal amino acid sequence of the blotted protein was determined and used for the construction of a degenerated and inosine-containing oligonucleotide primer for PCR experiments with cDNA transcribed from leaf mRNA. A 3-RACE experiment with this primer amplified a single band of 1500 bp that after sequencing showed an open reading frame of 382 amino acids corresponding to a protein of 43 kDa. The 5 end of the cDNA was amplified by a 5-RACE experiment and isolated as a 500 bp fragment. Sequencing of this DNA revealed an additional 65 amino acids at the N-terminus of the native protein that are attributed to a plastidial leader peptide. With appropriate primers derived from these sequences a full-length clone was amplified by PCR and sequenced. Comparison of the plastidial oleate desaturase with the homologous enzyme from cyanobacteria showed about 50% amino acid homology. Comparison with other desaturases revealed three histidine boxes with the general sequence HXXXH that are highly conserved in all membrane-bound desaturases. These boxes might be involved in metal ion complexation required for reduction of oxygen.     

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.     

Glycosylation in cardenolide biosynthesis

The glycosylation and deglycosylation of cardiac glycosides was investigated using cell suspension cultures and shoot cultures, both established from Digitalis lanata EHRH. plants, as well as isolated enzymes. Shoots were capable of glucosylating digitoxigenin, evatromonoside, digiproside, glucodigitoxigenin and digitoxin. Suspension cultured Digitalis cells glucosylated all the substrates mentioned but digiproside, whereas the UDP-glucosedependent cardinolide glucosyltransferase isolated from that source did not accept digitoxigenin and digiproside as substrates. It is concluded that at least three different glucosyltransferases are involved in cardiac glycoside formation in Digitalis. Similar experiments carried out with glucosylated cardenolides which were administered to cultured cells, shoots and a cardenolide -glucosidase isolated from young leaves revealed that at least two different glucosidases occur in Digitalis lanata, albeit in different tissues or during different phases of development. The biotransformation of glucoevatromonoside was investigated using unlabelled compound and [¹⁴C-glucose]-glucoevatromonoside synthesized enzymatically. After 7 d of incubation almost no radioactivity could be recovered from the cardenolide fraction, indicating that the terminal glucose of glucoevatromonoside was now incorporated into volatile, hydrophilic and insoluble compounds. Since, on the other hand, large amounts of cardenolides were found in the experiments with unlabelled glucoevatromonoside it is assumed that steady state or pool size regulation is achieved by the coordinated action of a cardenolide glucosidase and a glucosyltransferase.Abbreviations Acdox D-acetyldigitoxose - dgen digoxigenin - dox D-digitoxose - dten digitoxigenin - dtl D-digitalose - fuc D-fucose - gten gitoxigenin - qun D-quinovose - CGH cardenolide 16-O-glucohydrolase - DFT UDP-fucose:digitoxigenin 3-O-fucosyltransferase - DGT UDP-glucose:Digitoxin 16-O-glucosyltransferase - DQT UDP-quinovose:digitoxigenin 3-O-quinovosyltransferase     

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     

Efficiency of the tetracycline-dependent gene expression system: complete suppression and efficient induction of the rolB phenotype in transgenic plants

We have investigated the use of the tetracycline-dependent gene expression system to regenerate and propagate tobacco plants transformed with a gene whose product — when highly expressed — interferes with regeneration and/or further reproduction. Plants transformed with the Agrobacterium rhizogenes rolB gene under the control of the tetracycline-dependent expression system were phenotypically indistinguishable from wild type owing to efficient repression of the promoter. Induction of the rolB gene with tetracycline led to high-level expression of the rolB mRNA, which resulted in extremely stunted plants with necrotic and wrinkled leaves that did not develop a floral meristem. Upon cessation of tetracycline treatment healthy shoots developed even from severely affected meristems. Data on the dose response of the rolB phenotype as a function of tetracycline concentration demonstrate that the tetracycline-dependent gene expression system can be used to modulate the manifestation of a particular phenotype.     

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.     

Involvement of putrescine in the inductive rooting phase of poplar shoots raised in vitro

Micropropagated poplar shoots rooted 100% on a rooting medium (A) containing NAA, but they did not root in the absence of auxin (NA). Putrescine, but not spermidine and spermine, promoted rooting up to 42% when added to the NA medium. Cyclohexylamine (CHA), an inhibitor of spermine synthase, also promoted (up to 36%) rooting in the absence of auxin. The inhibitors of polyamine biosynthesis DFMA (α-difluoromethylarginine) and DFMO (α-difluoromethylomithine), aminoguanidine (AG) and methylglyoxal-bis-guanylhydrazone (MGBG), inhibited rooting when applied in the presence of auxin and had no effect in its absence.
The rooting inductive phase (in the presence of auxin) was determined by periodical transfer of shoots from A to NA medium, and by changes in peroxidase activity, to be 7 h. Putrescine (not spermidine and spermine) accumulated to a maximum during the inductive phase. Both putrescine and CHA promoted rooting on NA medium when applied during the first 7 h. In contrast DFMA and AG inhibited rooting during this period. The results point to the involvement of putrescine and its Δ¹-pyrroline pathway, in the inductive phase of rooting in poplar shoots.