Since a proper balance between anabolic and catabolic reactions is essential for all eukaryotes, the basic mechanisms for regulation of the energy and carbon metabolism have been conserved throughout evolution. The moss Physcomitrella patens, which belongs to one of the basal clades among land plants, has many unique properties that make it an excellent plant model system. We have used a yeast two-hybrid system to identify novel possible regulators or targets of the moss Snf1-related kinases, previously shown to regulate energy homeostasis. The function of the identified interactors PpSki1 and PpSki2 was analyzed in order to better understand the biological role of plant Snf1-related kinases. The recently completed genome sequence of Physcomitrella was used in a comparative approach to study to what extent key enzyme and gene families involved in transport and metabolism of sugars and in regulation of the energy and carbon metabolism are conserved between mosses and vascular plants. It has long been known that transformed DNA can replicate episomally in Physcomitrella. We have now shown that such DNA can be rescued back into E. coli. Surprisingly, we found that the original plasmid can be recovered from moss transformants obtained with circular DNA. Plasmids rescued from transformants obtained with linearized DNA had been repaired either by homologous recombination or by cohesive end re-ligation. These findings suggest that methods using shuttle plasmids are feasible in Physcomitrella. Hexokinase, a key enzyme in the carbon metabolism, catalyzes the first step in hexose metabolism, but is also involved in sugar sensing and signaling. We have now made an initial characterization of the complete hexokinase family in Physcomitrella which is encoded by 11 genes. Two new types of plant hexokinases, types C and D, were found in addition to the previously described types A and B.

The ability for an organism to sense and respond appropriately to its environment is often critical for survival. One mechanism for this is the inositol phosphate (InsP) signaling pathway. This work focuses on the role of InsP signaling in maintaining energy homeostasis in the plant. InsP signaling is connected to energy sensing in plants via a protein complex containing both the inositol polyphosphate 5-phosphatases (5PTase13) and the Sucrose non-Fermenting Related Kinase 1 (SnRK1). SnRK1 is considered a fuel gauge for the plant cell that senses energy status and reprograms growth appropriately. While the SnRK1.1 gene has been well studied, the role other SnRK1 isoforms play in energy or stress signaling is less well understood. This work examined the role of 3 SnRK1 isoforms in energy signaling, finding that SnRK1.1 and SnRK1.2 are regulated and function differently in Arabidopsis. The second part of this work focuses on the inositol pyrophosphates, which are a novel group of InsP signaling molecules containing diphosphate or triphosphate chains (i.e. PPx) attached to the inositol ring. These PPx-InsPs are emerging as critical players in the integration of cellular metabolism and stress signaling in non-plant eukaryotes. Most eukaryotes synthesize the precursor molecule, myo-inositol (1,2,3,4,5,6)-hexakisphosphate (InsP6), which can serve as a signaling molecule or as storage compound of inositol, phosphorus, and minerals. Even though plants produce huge amounts of InsP6 in seeds, almost no attention has been paid to whether PPx-InsPs exist in plants, and if so, what roles these molecules play. This work details the presence of PPx-InsPs in plants and delineates two Arabidopsis gene products (AtVip1 and AtVip2) capable of PP-InsP5 synthesis. We further examined the subcellular location of enzymes connected to PPx-InsP synthesis as well as the developmental and tissue specific patterns of expression of the genes that encode these enzymes. We localized the enzymes involved in InsP6 and PPx-InsP production to the nucleus and endoplasmic reticulum (ER). The subcellular compartmentalization of PPx-InsP signaling may be unique to plants. An increased understanding in the pathways involved in energy sensing and metabolic response may reveal novel strategies to improve crops for yield and viability in the future. / Ph. D.

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