Functional studies of plant hexokinases and development of genetic methods in the moss Physcomitrella patens

In the field of biology, model systems are frequently used for practical reasons. Model organisms possess several features that make them easy to work with in a laboratory setting. In addition they usually have a host of established genetic tools that have been developed by the research community. This thesis explores the potential of adapting methods and tools used in the yeast Saccharomyces cerevisiae to the plant model system Physcomitrella patens. We have studied the fate and integrity of plasmids transformed into Physcomitrella with the purpose of developing a working shuttle plasmids system in moss. Other methods from yeast genetics such as the use of auxotrophic strains would also be useful if adapted to the Physcomitrella model system. Experiments using Physcomitrella patens showed that a histidine auxotrophic moss strain can be complemented with the wild type gene on a plasmid and that the plasmid can be rescued back into E. coli. This indicates that shuttle plasmids, auxotrophic marker genes such as PpHIS3, cloning by complementation and perhaps even dosage suppressor screens could be used in Physcomitrella patens. Furthermore this thesis investigates the hexose phosphorylating enzyme hexokinase in Physcomitrella patens as well as in the charophyte alga Klebsormidium nitens. The characterization and localization studies performed on the eleven Physcomitrella hexokinases led to the discovery of two new types of plant hexokinases, the type C and type D hexokinases. In addition to these two new types, Physcomitrella contained several of the previously described type A and type B hexokinases. In addition to its enzymatic function, hexokinases in both plants and fungi have been implicated in glucose sensing and signaling. This thesis examines if hexokinase 1 of the microalga Klebsormidium nitens may play a similar role in glucose sensing as observed in higher plants. With this purpose in mind transgenic lines of the Arabidopsis thaliana glucose insensitive mutant gin2-1 expressing the Klebsormidium nitens hexokinase 1 were created. It was found that the K. nitens hexokinase can complement the glucose signaling defect in the gin2-1 mutant