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n 2011, two polytunnel greenhouse experiments were conducted on seed production farms, one under conventional conditions in the South-West (Rilland) and the other under organic conditions in the East (Voorst) of the Netherlands, to study transmission routes of Xanthomonas campestris pv. campestris (Xcc), that can result in seed infections of cauliflower plants (Brassica oleracea var. botrytis). Each experiment consisted of three plots. Before head formation, plants in the two outside plots were spray-inoculated with either Xcc strain IPO 3076 or IPO 3078, and plants in the central plot were sprayed with water. A colony of honeybees (Apis mellifera) was placed in each tunnel to provide pollination. At Rilland, incidences of black rot were low, and seed infections were not detected in a plating assay, although peduncles were infected in a high percentage of the plants. At Voorst, black rot symptoms developed in almost all Xcc-inoculated plants, resulting in plant losses. The incidence of plants with infected seeds in the plot inoculated with Xcc IPO 3076 was higher than in the two other plots. Identification of Xcc isolates by BOX-PCR revealed that Xcc IPO 3076 was transmitted to the other plots of the experiment. Xcc IPO 3078, however, was only recovered from plants inoculated with this strain. The routes of seed infection for Xcc and then transmission of the pathogen are discussed

Plants are regarded as a promising system for the production of heterologous proteins. However, little is known about the influence of plant physiology and plant development on the yield and quality of the heterologous proteins produced in plants. To investigate this, tobacco (Nicotiana tabacum cv Samsun NN) was transformed with a single construct that contained behind constitutive promotors the light- and heavy-chain genes of a mouse antibody. The in planta stability of the antibody was analyzed in transgenic plants that were grown under high and low irradiation at 15°C and 25°C. High-light conditions favored the production of biomass, of total soluble protein, and of antibody. The plants grown at 25°C developed faster and contained less antibody per amount of leaf tissue than the plants grown at 15°C. Both endogenous protein and antibody content showed a strong decline during leaf development. The heavy chains of the antibody underwent in planta degradation via relatively stable fragments. In vitro incubations of purified plantibody with leaf extracts of wild-type tobacco indicated the involvement of acidic proteases. It is interesting that the same antibody produced by mouse hybridoma cells exhibited higher stability in this in vitro assay. This may be explained by the assumption that the plant type ofN-glycosylation contributes less to the stability of the antibody than the mouse-type of N-glycosylation. The results of this study indicate that proteolytic degradation during plant development can be an important factor affecting yield and homogeneity of heterologous protein produced by transgenic plants.

Blackspot related pigments were partially purified from bruised tubers of two commercial potato cultivars (cv. Bildtstar and cv. Lady Rosetta). Chemical characterization showed that these pigments consisted of protein and a relatively small amount of covalently bound constituents. These polymeric structures absorbed light throughout the visible spectrum without any maximum. They did not contain eumelanin. Quinic acid was detectable in hydrolysates of the pigments from Bildtstar, but not in those of Lady Rosetta, which indicated that chlorogenic acid may take part in blackspot formation, but is not essential for the discoloration. The results support the hypothesis that blackspot pigments are products of non-regulated reactions between nucleophilic amino acid residues in proteins, and quinones, which are derived from endogenous substrates of polyphenol oxidase. This means that blackspot formation most probably takes place in disintegrated cells.

Seed and leaf treatments with natural compounds having a low risk profile (LRP) were evaluated for their potential to induce resistance in cabbage plants (Brassica oleracea) against Peronospora parasitica, causal organism of downy mildew. The selection of 34 LRP compounds comprised micronutrients, organic compounds such as proline, riboflavin, oligogalacturonides, aminolignosulfonates, bacterial lipopolysaccharides, and bacterial and fungal extracts. Treatments with the synthetic chemical inducers 2,6-dichloroisonicotinic acid (INA), d,l-s-aminobutyric acid, salicylic acid, benzothiadiazole and the fungicide Previcur™ were included as controls. After seed treatment a maximum reduction of 27% diseased leaf area was found with an extract of a Lysobacter strain, compared to a reduction of 99% for INA, the most effective synthetic inducer. Seed treatments with extracts of Pectobacterium carotovorum subsp. carotovorum, Bacillus macerans, Pseudomonas syringae, Streptomyces and Xanthomonas campestris strains also reduced downy mildew infection significantly. After leaf treatment, a maximum reduction of 85% was again found with the Lysobacter extract, compared to a reduction of 99% for INA, the most effective synthetic inducer. Leaf treatments with CuSO4 (=1 mM), MnCl2 (=10 mM), K2HPO4 (100 mM), and extracts of P. syringae, P. carotovorum subsp. carotovorum, Streptomyces, X. campestris and B. macerans strains also reduced the diseased leaf area, but CuSO4 was highly phytotoxic. For seed and leaf treatments with Lysobacter extract, proline, MnCl2 and INA the effect on the induction of chitinase and glucanase activity was tested, using two pathogenesis-related proteins as markers for induced resistance. For seed treatments only INA and for leaf treatments INA, proline and MnCl2 treatments resulted in increased activity of both enzymes. The rate of enzyme activity induced by INA was dependent on the time seeds were exposed to the compound. Highlights ? Seed treatments with isonicotinic acid protects Brassica seedlings from Peronospora infections. ? Treatments of seedlings with extracts of Lysobacter protects against Peronospora infections. ? Effect of seed treatments is dependent on the time of incubation with the elicitor

Four isoforms of strictosidine synthase (EC 4.3.3.2), which catalyses the stereospecific condensation of secologanin and tryptamine, were purified to h

Protein separation by hydrophobic interaction chromatography (HIC) depends on the differences in surface hydrophobicity of the constituent proteins and is hence complementary to chromatographic techniques based on the charge or the size of the macromolecule. This review summarises some of the recent developments in HIC supports and discusses the parameters that must be considered in designing an efficient chromatographic system. The application of HIC to the purification of enzymes of secondary compound biosynthesis that cannot be obtained in pure form by size-exclusion and ion-exchange techniques alone is exemplified by reference to recent studies on tryptophan decarboxylase, strictosidine synthase and geraniol-10-hydroxylase.

Plants are regarded as a promising system for the production of heterologous proteins. However, little is known about the influence of plant development and growth conditions on N-linked glycosylation. To investigate this, transgenic tobacco (Nicotiana tabacum cv Samsun NN) plants expressing a mouse immunoglobulin G antibody (MGR48) were grown in climate rooms under four different climate conditions, i.e. at 15°C and 25°C and at either low or high light conditions. N-glycans on plantibodies and soluble endogenous proteins were analyzed with matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS). Antibodies isolated from young leaves have a relatively high amount of high- mannose glycans compared with antibodies from older leaves, which contain more terminal N-acetylglucosamine. Senescence was shown to affect the glycosylation profile of endogenous proteins. The relative amount of N-glycans without terminalN-acetylglucosamine increased with leaf age. Major differences were observed between glycan structures on endogenous proteins versus those on antibodies, probably to be attributed to their subcellular localization. The relatively high percentage of antibodyN-glycan lacking both xylose and fucose is interesting.

Plant-specificN-glycosylation can represent an important limitation for the use of recombinant glycoproteins of mammalian origin produced by transgenic plants. Comparison of plant and mammalianN-glycan biosynthesis indicates that β1,4-galactosyltransferase is the most important enzyme that is missing for conversion of typical plantN-glycans into mammalian-likeN-glycans. Here, the stable expression of human β1,4-galactosyltransferase in tobacco plants is described. Proteins isolated from transgenic tobacco plants expressing the mammalian enzyme bearN-glycans, of which about 15% exhibit terminal β1,4-galactose residues in addition to the specific plantN-glycan epitopes. The results indicate that the human enzyme is fully functional and localizes correctly in the Golgi apparatus. Despite the fact that through the modified glycosylation machinery numerous proteins have acquired unusualN-glycans with terminal β1,4-galactose residues, no obvious changes in the physiology of the transgenic plants are observed, and the feature is inheritable. The crossing of a tobacco plant expressing human β1,4-galactosyltransferase with a plant expressing the heavy and light chains of a mouse antibody results in the expression of a plantibody that exhibits partially galactosylatedN-glycans (30%), which is approximately as abundant as when the same antibody is produced by hybridoma cells. These results are a major step in thein plantaengineering of theN-glycosylation of recombinant antibodies.

Partially purified blackspot pigments from potato tubers (Solanum tuberosum L.) of two commercial cultivars were subjected to a microassay for melanin, which consisted of specific chemical degradation and subsequent HPLC analysis. Permanganate oxidation yielded pyrrole-2,3,5-tricarboxylic acid, whereas hydrolysis in hydriodic acid liberated aminohydroxyphenylalanine isomers. These results indicate that the polymeric pigments, which have previously been found to contain a protein matrix, carry crosslinked 5,6-dihydroxyindole-2-carboxylic acid and benzothiazine units. This leads to the conclusion that free tyrosine and free cysteine are incorporated in the proteinaceous pigments via the polyphenol oxidase catalysed pathway of melanogenesis in the process of blackspot formation. The findings are in accordance with the hypothesis that the process of blackspot formation is a non-regulated cascade of reactions in disintegrated tuber cells, rather than a finely tuned biosynthesis.

In a study of six potato varieties (Solanum tuberosum L.), which were grown under two different fertilization regimes, the biochemical potential of tuber tissue to synthesize blackspot pigments was distinguished from the actual blackspot susceptibility exhibited after impact application. Blackspot susceptibility and biochemical potential for pigment synthesis were not correlated, which supports the hypothesis that the extent to which the blackspot potential is actually being used is mediated by the sensitivity to cell decompartmentation. Quantification of polyphenol oxidase (PPO), soluble protein, and endogenous PPO substrates demonstrated that the content of free tyrosine is the predominant determinant for the biochemical potential for blackspot synthesis. PPO was not a limiting factor for pigment synthesis, even if PPO content was relatively low. It was therefore concluded that manipulation of PPO activity may offer a solution to the problem of blackspot formation only if it leads to a virtually complet...

The subcellular localization of tryptophan decarboxylase, strictosidine synthase and strictosidine glucosidase in suspension cultured cells of Catharanthus roseus (L.) G. Don and Tabernaemontana divaricata (L.) R. Br. ex Roem. et Schult, was investigated. It was found that tryptophan decarboxylase is an extra-vacuolar enzyme, whereas strictosidine synthase is active inside the vacuole. Strong indications were obtained for the localization of strictosidine glucosidase on the outside of the tonoplast. The results suggest that tryptamine is transported into the vacuole where it is condensed with secologanin to form strictosidine, and that strictosidine passes the tonoplast and is subsequently hydrolysed outside the vacuole.