Group Members & Contact Information
Research in my group is focused on elucidating mechanisms leading to formation of nucleotide- sugars (especially UDP-sugars), which are direct precursors to polysaccharides (e.g. sucrose, cellulose, hemicellulose). The function of relevant enzymes is elucidated both in vivo, using transgenic plants, and by studying purified proteins. Structure/ function properties of key enzymes are investigated, using site-directed mutagenesis, domain deletion approaches, specific inhibitors, etc. In another project, involving studies on sugar effects on global gene expression, we dissect signaling roles of a given sugar from its metabolic function, identifying genes specifically regulated by a given sugar, and using bioinformatics/ functional genomics to define cis motifs responsible for gene regulation.

UDP-sugars are used in hundreds of glycosylation reactions (e.g. those forming sucrose, cell wall polysaccharides, etc.), and they represent the most important precursors for biomass production in nature. Thus, mechanisms of UDP-sugar formation, together with selected glycosyltransferases, may represent primary targets for biotechnological approaches aiming at increased biomass production for needs of agriculture, forestry and other industries. We are studying both genes and proteins responsible for UDP-sugar production, including those for UDP-Glc pyrophosphorylase (UGPase), sucrose synthase and, recently, UDP-sugar pyrophosphorylase (USPase) and UDP-N-acetylglucosamine pyrophosphorylase (UAGPase). Since sugars are essential for UDP-sugar synthesis, we also investigate sugar-mediated regulation of plant gene expression, using both cell culture and whole plants.

In vivo roles and function/structure studies on pyrophosphorylases
We are using transgenic plants with impaired expression of a given protein to elucidate its physiological role. Parameters studied include growth/development of transgenics, metabolite contents, physiological properties, co-expressed gene networks, etc. At the protein level, purified plant pyrophosphorylases are investigated with respect to molecular determinants of their substrate specificity and regulation, including effects of oligomer- ization and post-translational modifications.The enzymes differ in specificity for sugar and nucleotide portions of their substrates, nd may be involved in distinct metabolic pathways. Properties of the enzymes are modified via site-directed mutagenesis and domain swapping. We are also searching for specific inhibitors of pyrophosphorylases, using a chemical library developed in Chemistry Dept. at Umeå Univ. Inhibitors will be used to study the role of the target protein in selected physiological/ developmental processes in plant tissues/ cells (reverse chemi- cal-genetics approach) and, possibly, as potential drugs against human protozoan pathogens (e.g. Leishmania, Trypanosoma).

Central role of UDP-glucose in primary sugar metabolism.	Structures of plant UDP-sugar producing pyrophosphorylases (Kleczkowski et al. 2011).

Sugar signaling and regulation of gene expression
In this project, we aim to understand a sugar-dependent regu- lation of gene expression by characterizing target genes and the underlying mechanism(s) of the regulation, using both an in vitro Arabidopsis cell culture and whole plants. Using the cell culture, we study in detail the response of cells to short-term exposures to low concentrations of different sugar species (e.g. Suc, Glc, and Fru), quantifying the sugar-dependent changes in gene expres- sion by microarray and qPCR approaches. Potential signaling pathways that are involved in the regulation of identified early responsive target genes are then further investigated by the use of known signaling mutants of Arabidopsis, e.g. hexokinase lines. To understand the mechanism(s) underlying the regulation, we study the sugar-dependent expression of promoter::reporter genes. The reporter constructs contain defined cis-regulatory elements in their promoters, which are both synthetic as well as derived from native genes,and their expression is analyzed both in stably transformed cell cultures and whole plants.
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