Correlation of resistance and H2O2 production in Ulmus pumila and Ulmus campestris cell suspension cultures inoculated with Ophiostoma novo-ulmi.

The Dutch elm disease (DED) pathogen Ophiostoma novo-ulmi Buissm. elicited the production of H2O2 in cell suspension cultures of the resistant species Ulmus pumila L. This response was not observed in suspensions of the susceptible elm U. campestris Mill. H2O2 production started after a lag time of 30-40 min following inoculation, peaked between 4 and 6 h and lasted up to 24 h. Treatment of the suspensions with exogenously added H2O2 did not cause accumulation of the sesquiterpene phytoalexins mansonones nor of the coumarin scopoletin. Spore germination and growth of O. novo-ulmi were significantly delayed with different amounts of H2O2 (0.1-1 mM). These results suggest that H2O2 production is an inducible defence response which may contribute to DED resistance by delaying the growth of the pathogen at the earliest stages of infection. Whether H2O2 is involved in other elm defence responses to the pathogen is presently unknown, but its production seems to be an independent event from phytoalexin formation.

Proteome alterations monitored by DIGE analysis in Silybum marianum cell cultures elicited with methyl jasmonate and methyl B cyclodextrin.

Elicitation with methyl jasmonate (MeJA) or/and cyclodextrin (CD) strongly induced silymarin (Sm) accumulation in suspensions of Silybum marianum, with most of Sm isomers being detected in the culture medium. This induction provides a model platform to characterize the regulation of flavonolignan accumulation and release in response to elicitors and, with this aim, changes in the S. marianum cell proteome were investigated. The DIGE technique was used to detect statistically significant changes in the cell's proteome. A total number of 1269 unique spots were detected, 67 of which were de-regulated upon elicitation. Nineteen spots were identified by nLC-MS/MS database search analysis. Identified proteins belong to a few categories, including metabolism, stress and defense responses and transport processes. The most abundant group was represented by pathogenesis-related (PR) proteins and heat shock proteins. Two proteins related to transport process were identified and both were upregulated by elicitation. One was identified as Ras-related protein Rab11C of the Rab family of small ATPase superfamily. A second protein was identified as an ABC transporter. Some of the identified proteins are discussed with respect to their putative role in the extracellular flavonolignan accumulation in S. marianum cultures. BIOLOGICAL SIGNIFICANCE: Most approaches to increase secondary metabolite yields using plant cell cultures have been focused on the optimization of its biosynthesis. The study of other post biosynthetic events, like chemical or enzymatic modifications, transport, storage/secretion and catabolism/degradation are also biotechnologically relevant. Secretion is of particular interest since if cell cultures are to be used routinely for the commercial production, they must release the targeted metabolites into the extracellular medium. Elicitor-induced silymarin accumulation and release in S. marianum cell cultures provide a responsive model system to profile both alterations in proteins related to monolignol/flavonoid biosynthesis and to identify potential systems involved in secretion of secondary metabolites. The proteomic approach undertaken in this work has permitted identify some of the events occurring in elicited S. marianum cell cultures. One attainment of this study is that a vesicular transport mechanism could be involved in the release of this class of secondary metabolites to the extracellular compartment. This finding forms a baseline for future research on a non-sequenced medicinal plant S. marianum at molecular level.

Biotransformation of (-)-codeinone to (-)-codeine byPapaver somniferum cells immobilized in reticulate polyurethane foam.

Papaver somniferum cells immobilized in reticulate-polyurethane foam biotransformed (-)-codeinone to (-)-codeine. A biotransformation ratio of 79% was found in immobilized cells whereas a ratio of 57% was found in suspended cells. Of the total amount of codeine formed only 12.2% was detected inside the cells, most of the product (87.3%) being released into the medium. When immobilized cells were cultivated in the absence of nitrate, only 40% of the cells remained alive and the biotransformation of codeinone was strongly reduced. When orthophosphate was omitted from the growth medium a bioconversion ratio of 86% was achieved.

Calcium restriction induces cardenolide accumulation in cell suspension cultures of Digitalis thapsi L.

The removal of calcium ions from Murashige and Skoog culture medium induced a marked increase in the accumulation of cardenolides in cell suspension cultures of Digitalis thapsi. Cell viability was not affected although growth was slightly reduced. Strontium ions could substitute for calcium in inhibiting cardenolide production, this effect of calcium being reversed by the addition of LaCl3 or ethyleneglycol-bis-(ß-aminoethyl ether)-N,N'-tetraacetic acid. The results suggest that calcium, apart from its general effects on growth, may play a role in the regulation of cardenolide metabolism in a concentration dependent manner.