Developing seeds: In 2002, graduate student Jim Blonde made the groundbreaking discovery of high and low molecular weight PEP carboxylase isoforms in developing castor oil seeds (remarkably analogous to our previously characterized green algal ‘Class 1’ and ‘Class 2’ PEP carboxylase isoforms). These isoforms display profound differences in their respective physical and kinetic/allosteric properties, and response to reversible in vivo phosphorylation. They are hypothesized to play a key role in controlling the partitioning of imported photosynthate (sucrose) to storage protein vs. storage oil synthesis in the developing seeds. This research provides the only evidence to date that demonstrates a probable function for the enigmatic ‘bacterial-type’ PEP carboxylase isozyme of vascular plants, initially identified through genome sequencing initiatives. We are also interested in the corresponding PEP carboxylase protein kinases and protein phosphatases that mediate the reversible in vivo phosphorylation of castor seed PEP carboxylases.
Germinating seeds: In March 2008 Glen Uhrig demonstrated that the onset of castor seed germination is accompanied by monoubiquitination of 50% of PEP carboxylase's subunits at a conserved lysine residue, resulting in stable formation of an unusual Class-1 PEPC heterotetramer having altered kinetic/regulatory properties. Polyubiquitination is well known to 'tag' damaged proteins for their proteolytic degradation. However, many non-destructive functions for protein monoubiquitination in yeast and animals have recently emerged, particularly to mediate protein-protein interactions and protein localization to help control processes such as endocytosis, transcription and translation, and signal transduction. UB-related pathways are also believed to comprise over 6% of the Arabidopsis or rice proteomes with thousands of different proteins being probable targets. An important goal in the post-genome era will be the characterization of the monoubiquitinated proteome of plant cells, the UB-binding domain proteins that they interact with, and the influence of this post-translational modification on target protein localization, protein:protein interactions, and functional properties. Although germinating castor seed PEPC carboxylase provides the 1st example in nature that in vivo monoubiquitination of a metabolic enzyme can occur, additional research is underway to assess: (1) what UB-binding domain proteins interact with this enzyme in vivo, (2) whether this modification contributes to formation of a glycolytic metabolon (multienzyme complex) while minimizing futile cycling between PEP carboxylase and PEP carboxykinase (a highly active gluconeogenic enzyme in germinating oilseeds), & (3) the UB E3 ligase and related signaling pathway that result in PEP carboxylase's monoubiquitination early in the germination process.
Model illustrating the biochemical complexity of developing vs. germinated castor oil seed (COS) PEP carboxylase (PEPC).
In developing castor beans the plant-type PEPC RcPPC3 exists as: (1) a typical Class-1 PEPC homotetramer which is activated in vivo by sucrose dependent phosphorylation of 50% of its p107 subunits (at Serine-11), & (2) part of the novel allosterically-desensitized Class-2 PEPC hetero-octameric complex with the multi-site phosphorylated bacterial-type PEPC RcPPC4 (p118) subunits. COS germination is accompanied by increases in RcPpc3 gene expression, PEPC activity and amount, and monoubiquitination of 50% of p107 subunits at Lysine-628 to form the novel Class-1 PEPC p110:p107 heterotetramer.
Our recent research in this area has included: (1) the complete purification and detailed biochemical and genetic characterization of the predominant Pi starvation upregulated intra- and extracellular purple acid phosphatases of Arabidopsis cell cultures and seedlings, and (2) demonstrating that the PEP carboxylase isozyme AtPPC1 is simultaneously induced and activated by protein kinase-mediated phosphorylation in Pi starved Arabidopsis. We have also been using a proteomics approach to discover novel phosphate starvation inducible and differentially phosphorylated proteins of Pi-deprived Arabidopsis.
Model illustrating alternative pathways of cytosolic glycolysis, miETC, and tonoplast H+-pumping processes (indicated by bold arrows) that may facilitate respiration and vacuolar pH maintenance by Pi-deprived plant cells.
These bypasses negate any dependence of respiration and tonoplast H+-pumping on adenylates and/or Pi, the levels of which become markedly depressed during severe Pi starvation. Organic acids produced by PEP carboxylase may also be excreted by roots to increase the availability of mineral bound Pi. A key component of this model is the critical secondary role played by ‘metabolic Pi recycling systems’ during Pi deprivation.
THE MYTH OF PHOSPHITE FERTILIZERS. Over the past 25 years, a reduced form of Pi known as phosphite (Phi) has been widely used to improve the yield of many crops. Phi’s use in agriculture is highly controversial since it is being marketed both as a crop fungicide and as a superior source of crop phosphorus nutrition (relative to Pi). In the mid-1990s we began to assess the influence of Phi on plant growth and metabolism. We discovered that low Phi concentrations are highly phytotoxic to Pi-starved, but not Pi-sufficient plants (and yeast) because Phi blocks the upregulation of Pi-starvation inducible enzymes and Pi transporters needed for plant acclimation to suboptimal Pi nutrition. Several top labs in the field of plant P-nutrition (including K.G. Raghothama at Univ. of Purdue, Steffen Abel at U.C. Davis, Deb Allan & Carroll Vance at Univ. of Minnesota ) have since employed Phi as a tool to help dissect plant Pi sensing pathways. The data in plants and yeast are consistent with the hypothesis that cell sensors and/or signaling components involved in the coordinated response to Pi-stress actually perceive Phi as Pi even though Phi is not metabolized. Nevertheless, farmers throughout the world are applying Phi formulations marketed as a "super P fertilizer" rather than as a fungicide; e.g., Nutri-Phite, Phosgard, & Nutramix, etc. Agrochemical companies selling 'phosphite fertilizer' products appear to be avoiding the substantial expense and time associated with registering an agricultural fungicide (by labeling their Phi products as a "P fertilizer"). As a result of our phosphite research, The Fertilizer Section of the Canadian Food Inspection Agency (CFIA) recently banned sales of so-called ‘phosphite fertilizer’ products in Canada (see CFIA trade memorandum T-4-12 on "Requirements for Phosphite and Phosphorous Acid Materials Represented for Use as Fertilizers" at http://www.inspection.gc.ca/english/plaveg/fereng/tmemo/t-4-121e.shtml )
