Inducible expression of Pisum sativum xyloglucan fucosyltransferase in the pea root cap meristem, and effects of antisense mRNA expression on root cap cell wall structural integrity

Mitosis and cell wall synthesis in the legume root cap meristem can be induced and synchronized by the nondestructive removal of border cells from the cap periphery. Newly synthesized cells can be examined microscopically as they differentiate progressively during cap development, and ultimately detach as a new population of border cells. This system was used to demonstrate that Pisum sativum L. fucosyl transferase (PsFut1) mRNA expression is strongly expressed in root meristematic tissues, and is induced >2-fold during a 5-h period when mitosis in the root cap meristem is increased. Expression of PsFut1 antisense mRNA in pea hairy roots under the control of the CaMV35S promoter, which exhibits meristem localized expression in pea root caps, resulted in a 50–60% reduction in meristem localized endogenous PsFut1 mRNA expression measured using whole mount in situ hybridization. Changes in gross levels of cell wall fucosylated xyloglucan were not detected, but altered surface localization patterns were detected using whole mount immunolocalization with CCRC-M1, an antibody that recognizes fucosylated xyloglucan. Emerging hairy roots expressing antisense PsFut1 mRNA appeared normal macroscopically but scanning electron microscopy of tissues with altered CCRC-M1 localization patterns revealed wrinkled, collapsed cell surfaces. As individual border cells separated from the cap periphery, cell death occurred in correlation with extrusion of cellular contents through breaks in the wall

Connected nucleophilic substitution-Claisen rearrangement in flow : analysis for kilo-lab process solutions with orthogonality

The two-step synthesis of phenol to 2-allylphenol in micro flow is investigated. This synthesis involves a nucleophilic substitution (SN2) reaction of phenol with allyl bromide towards allyl phenyl ether and the thermal Claisen rearrangement of allyl phenyl ether to 2-allylphenol. This carbon–carbon bond forming reaction route would provide a valuable path towards complex molecules. Flow cascades have turned into a powerful approach to provide chemical diversity (process-design intensification). This is enabled by chemical intensification of the Claisen rearrangement in micro flow, by reducing the reaction time to minutes without the need of a catalyst. While both individual reaction steps have been optimized separately in earlier research, an initial directly connected two-step synthesis gave low selectivity. Accordingly, the main topic investigated is how to achieve orthogonality in case of reagent mismatch between the two reactions. First, four flow process protocols using three different kinds of in-flow separation and one kinetic approach, are developed at laboratory scale. From there, process design sheets for kilolab processing set-ups of the suited approaches are developed which shed first light on their industrial practicality. In particular, it has been found that the main causes for the drop in selectivity are the presence of the base DBU and the reactant allyl bromide during the Claisen rearrangement. Three of the four investigated separation approaches demonstrated the ability to improve the overall yield – acid–base extraction, acid absorption by using ion exchange resin, using heterogeneous base, and dilution as kinetic approach. Finally, for every option, the proposed respective production set-up, anticipated advantages and drawbacks are given to facilitate a decision. Keywords Flow chemistry; Microreactors; Claisen rearrangement; Continuous process desig