Microfluidic capillary zone electrophoresis mass spectrometry analysis of alkaloids in Lobelia cardinalis transgenic and mutant plant cell cultures.

Application of a microfluidic CE* device for CZE-MS allows for fast, rapid, and in-depth analysis of large sample sets. This microfluidic CZE-MS device, the 908 Devices ZipChip, involves minimal sample preparation and is ideal for small cation analytes, such as alkaloids. Here, we evaluated the microfluidic device for the analysis of alkaloids from Lobelia cardinalis hairy root cultures. Extracts from wild-type, transgenic, and selected mutant plant cultures were analyzed and data batch processed using the mass spectral processing software MZmine2 and the statistical software Prism 8. In total 139 features were detected as baseline resolved peaks via the MZmine2 software optimized for the electrophoretic separations. Statistically significant differences in the relative abundance of the primary alkaloid lobinaline (C27 H34 N2 ), along with several putative "lobinaline-like" molecules were observed utilizing this approach. Additionally, a method for performing both targeted and untargeted MS/MS experiments using the microfluidic device was developed and evaluated. Coupling data-processing software with CZE-MS data acquisition has enabled comprehensive metabolomic profiles from plant cell cultures to be constructed within a single working day.

Target-directed discovery and production of pharmaceuticals in transgenic mutant plant cells.

Plants are a source of complex bioactive compounds, with value as pharmaceuticals, or leads for synthetic modification. Many of these secondary metabolites have evolved as defenses against competing organisms and their pharmaceutical value is "accidental", resulting from homology between target proteins in these competitors, and human molecular therapeutic targets. Here we show that it is possible to use mutation and selection of plant cells to re-direct their "evolution" toward metabolites that interact with the therapeutic target proteins themselves. This is achieved by expressing the human target protein in plant cells, and selecting mutants for survival based on the interaction of their metabolome with this target. This report describes the successful evolution of hairy root cultures of a Lobelia species toward increased biosynthesis of metabolites that inhibit the human dopamine transporter protein. Many of the resulting selected mutants are overproducing the active metabolite found in the wild-type plant, but others overproduce active metabolites that are not readily detectable in non-mutants. This technology can access the whole genomic capability of a plant species to biosynthesize metabolites with a specific target. It has potential value as a novel platform for plant drug discovery and production, or as a means of optimizing the therapeutic value of medicinal plant extracts.

Iron plaques improve the oxygen supply to root meristems of the freshwater plant, Lobelia dortmanna.

* High radial oxygen loss (ROL) from roots of aquatic plants to reduced sediments is thought to deplete the roots of oxygen and restrict the distribution of those species unable to form a barrier to oxygen loss. Metal precipitates with high iron content (Fe-plaques) frequently form on roots of aquatic plants and could create such a diffusion barrier, thereby diverting a larger proportion of downward oxygen transport to the root meristems. * To investigate whether Fe-plaques form a barrier to oxygen loss, ROL and internal oxygen concentrations were measured along the length of roots of the freshwater plant Lobelia dortmanna using platinum sleeve electrodes and Clark-type microelectrodes. * Measurements showed that ROL was indeed lower from roots with Fe-plaques than roots without plaques and that ROL declined gradually with thicker iron coating on roots. The low ROL was caused by low diffusion coefficients through root walls with Fe-plaques resulting in higher internal oxygen concentrations in the root lacunae. * By diverting a larger proportion of downward oxygen transport to root meristems in L. dortmanna, the presence of Fe-plaques should diminish root anoxia and improve survival in reduced sediments.