In Planta - Complementation of Clavibacter Michiganensis Subsp. sepedonicus Strains Deficient in Cellulase Production or HR Induction Restores Virulence

Clavibacter michiganensis subsp. sepedonicus, a Gram positive bacterium that causes bacterial ring rot of potato, was studied in eggplant, an alternate host, using strains that differed in phenotype. Two factors affecting virulence, the ability to induce a hypersensitive response (HR) and cellulase production, were studied. A plasmid-free isolate of C. michiganensis subsp. sepedonicus that causes HR on tobacco but is unable to produce cellulase multiplied efficiently in planta, but caused only weak symptoms. In contrast, a strain that is unable to induce HR on tobacco but produces cellulase was impaired in the ability to multiply in the host and caused no symptoms. When the two non-virulent strains were coinoculated into eggplants, typical disease symptoms developed. This enhancement was not due to formation of a new phenotype or significant increases in population density of either of the strains. Our results suggest that both cellulase production and the ability to induce HR are required for a successful infection process and disease induction by C. michiganensis subsp. sepedonicus. Our results additionally suggest that the ability to induce HR on non-host plants is required for multiplication in the host plant, whereas cellulase expression is necessary for induction of disease symptoms.     

Mitotic Waves in Laticifers of Euphorbia marginata

A successive pattern of nuclear divisions that result in mitotic waves has been observed within the coenocytic nonarticulated laticifers of embryos of Euphorbia marginata Pursh. These waves originate independently in the cotyledonary or hypocotyl portion of the laticifer and exhibit uni-or bidirectional movement at variable velocities. Individual nuclei or groups of neighoring nuclei in a laticifer were observed in a sequence of mitotic stages ranging from prophase to telophase; division activity varied with individual laticifers in an embryo. Two mitotic patterns were apparent in the embryo: a random pattern associated with various cells in the meristematic area, and a successive pattern restricted to the laticifer. A substance, synthesized by and restricted to the laticifer, may be associated with this mitotic pattern.     

Light sources with different spectra affect root and mycorrhiza formation in Scots pine in vitro

We studied the effects of broad-spectrum light quality on the interaction between the ectomycorrhizal fungus Pisolithus tinctorius (Pers.) Coker and Couch and Scots pine (Pinus sylvestris L.) seedlings and hypocotyl cuttings cultured in vitro. The light sources were cool white (CW), warm white (WW) and red-rich daylight (RD) fluorescent lamps. Inoculation with P. tinctorius enhanced adventitious root formation of the cuttings in all light treatments. Rooting of the inoculated cuttings was highest in WW light (89%), followed by CW (73%) and RD light (66%). During 6 weeks of in vitro culture, rooted cuttings formed only a few lateral roots. The fungus grew over lateral roots, but the Hartig net was absent in all light treatments. In non-inoculated cuttings, neither root formation nor subsequent root growth was affected by light quality. In the seedling experiment, inoculation in the WW treatment resulted in a significantly (P < 0.05) greater number of lateral roots than inoculation in the RD treatment. The percentage of lateral roots covered with fungal hyphae was also highest in WW light (62%), followed by CW (50%) and RD (27%) light. A similar pattern was observed in the intensity of Hartig net formation. We conclude that effects of broad-spectrum light quality on the ectomycorrhizal fungus-root interaction are dependent on the developmental stage of the root.     

Polymerisation of added coniferyl alcohol by inherent xylem peroxidases and its effect on fungal decay resistance of Norway spruce

It is known that active peroxidase isozymes exist in mature wood of Norway spruce (Picea abies L. Karst.) and that they remain active for years and are found even in the heartwood (in Scots pine), where all cellular activity has ceased. This peroxidase activity was utilised in the impregnation of wood blocks with a natural monolignol, coniferyl alcohol and hydrogen peroxide. The hypothesis was that the internal wood peroxidases would oxidise the added monolignol and bind it stably into the cell wall matrix, which could hinder fungal decay. Since coniferyl alcohol is not very soluble in water, the impregnation was done under vacuum with an acetone–water solution containing 10% coniferyl alcohol and 0.4 mM H₂O₂ at room temperature (ca. 0.02 g of coniferyl alcohol was added to 1 g of wood). After impregnation, dimers of coniferyl alcohol and free coniferyl alcohol were found in acetone extracts with GC–MS analysis. Penetration of coniferyl alcohol and non-extractable reaction products were studied from the wood blocks with FTIR PAS technique. The wood samples treated were also subjected to a fungal decay test with Coriolus versicolor. This treatment hindered fungal decay in a 60-day experiment and led to a dry weight loss of 8.8% in comparison with 19.9% in the control. The reactions of coniferyl alcohol and H₂O₂ in the presence of peroxidases are discussed as well as the use of monolignols to increase wood decay resistance.     

Protein-lipid interactions during liposome oxidation with added anthocyanin and other phenolic compounds

Oxidation of bovine serum albumin, casein, and lactalbumin and the effect of different procyanidins, anthocyanins, and their aglycons (10 and 20 microM) on lactalbumin oxidation were investigated in a liposome system. Samples were incubated in the dark at 37 degrees C with copper, and the extent of oxidation was measured by determining the loss of tryptophan fluorescence and the formation of protein carbonyls, conjugated diene hydroperoxides, and hexanal. The correlation between different protein and lipid oxidation measurements was good and statistically significant. Casein was the most stable protein in the liposome model, and it was also the best inhibitor of liposome oxidation. All tested anthocyanins and other phenolic compounds inhibited both lipid and protein oxidation. There were no systematic differences with anthocyanins and their aglycons in relation to the concentrations used or glycosylation with either glucose or rutinose. Procyanidins B1 and B2 and ellagic acid were potentially better antioxidants than anthocyanins due to their several hydroxyl groups as measured by both protein and lipid oxidation. In conclusion, oxidative deterioration of liposomes due to protein-lipid interaction is inhibited by anthocyanins, procyanidins, and ellagitannin present, for example, in berries.     

Identification and quantification of phenolic compounds in berries of Fragaria and Rubus species (family Rosaceae)

High-performance liquid chromatography combined with diode array and electrospray ionization mass spectrometric detection was used to study soluble and insoluble forms of phenolic compounds in strawberries, raspberries (red and yellow cultivated and red wild), arctic bramble, and cloudberries. Hydroxycinnamic acids were present as free forms in cloudberries and mainly as sugar esters in the other berries. Quercetin 3-glucuronide was the typical flavonol glycoside in all of the berries studied. The composition of the predominant anthocyanins can be used to distinguish the studied red Rubus species from each other since cyanidin was glycosylated typically with 3-sophorose (56%) in cultivated red raspberry, with 3-sophorose (30%) and 3-glucose (27%) in wild red raspberry, and with 3-rutinose (80%) in arctic bramble. Ellagic acid was present as free and glycosylated forms and as ellagitannins of varying degrees of polymerization. Comparable levels of ellagitannins were obtained by the analysis of soluble ellagitannins as gallic acid equivalents and by the analysis of ellagic acid equivalents released by acid hydrolysis of the extracts.     

Transglutaminase catalyzed cross-linking of sodium caseinate improves oxidative stability of flaxseed oil emulsion

Sodium caseinate was modified by transglutaminase catalyzed cross-linking reaction prior to the emulsification process in order to study the effect of cross-linking on the oxidative stability of protein stabilized emulsions. The extent of the cross-linking catalyzed by different dosages of transglutaminase was investigated by following the ammonia production during the reaction and using SDS-PAGE gel. O/W emulsions prepared with the cross-linked and non-cross-linked sodium caseinates were stored for 30 days under the same conditions. Peroxide value measurement, oxygen consumption measurement, and headspace gas chromatography analysis were used to study the oxidative stability of the emulsions. The emulsion made of the cross-linked sodium caseinate showed an improved oxidative stability with reduced formation of fatty acid hydroperoxides and volatiles and a longer period of low rate oxygen consumption. The improving effect of transglutaminase catalyzed cross-linking could be most likely attributed to the enhanced physical stability of the interfacial protein layer against competitive adsorption by oil oxidation products.     

Charge modifications to improve the emulsifying properties of whey protein isolate

Whey protein isolate was modified by ethylene diamine in order to shift its isoelectric point to an alkaline pH. The extent of the modification was studied using SDS-PAGE and MALDI-TOF mass spectrometry. The modified whey proteins were used as an emulsifier to stabilize oil-in-water emulsions at acidic and neutral pH ranges, and their emulsifying properties were compared with that of the unmodified whey proteins and with the previously studied ethylene diamine modified sodium caseinate. The emulsifying activity of the modified whey proteins was similar to that of the unmodified ones, but the stability of an emulsion at pH 5 was significantly improved after the modification. Charge and coverage of droplet surface and the displacement of the interfacial proteins by surfactant Tween 20 were further studied as a function of pH. As compared with the unmodified whey proteins, the modified ones were proven to cover the interface more efficiently with extensive surface charge at pH 5, although the interfacial layer was less resistant to the surfactant displacement.