The major focus in the Huber laboratory is on identification of biological mechanisms that regulate important plant processes and impact growth and development. For example, sucrose synthesis and utilization; nitrate assimilation; and protein oil formation in developing soybean seeds. Studies include several levels but most work is focused on post-translational mechanisms. One such mechanism that plays an important role in the regulation of enzyme activity, but is still poorly understood in plant systems, is reversible protein phosphorylation. Each of the processes we are studying involves at least one enzyme that is phosphorylated.
Current projects in the lab include the following:
Control of Nitrate Assimilation
The enzyme nitrate reductase (NR) catalyzes the first step in the important process of nitrate assimilation, and is controlled at multiple levels. The activity of NR is regulated at the post-translational level by phosphorylation of a serine residue (Ser 543 in the hinge 1 region of spinach NR), followed by binding of a "14-3-3 protein" to form an inactive complex. Recent results also suggest that 14-3-3 proteins can also affect NR activity indirectly by reducing the proteolytic degradation of the NR protein in vivo. Our interest lies in the factors that govern the phosphorylation status of NR, as well as those that may control the ability of 14-3-3 proteins to bind to target proteins such as NR. For example, the 14-3-3s require an activator, such as a divalent cation, in order to bind to the target protein. A loop region common to all 14-3-3s has been identified as the divalent-cation binding site. Interestingly, polyamines (organic polycations) can also bind to the divalent-cation binding site and we are speculating that interaction with 14-3-3 proteins may be one basis for the action of polyamines as plant growth regulators. The activation by cations involves, at least in part, a conformational change in the C-terminus of the 14-3-3 protein, which functions as an "autoinhibitor". We are currently producing transgenic Arabidopsis plants expressing 14-3-3 proteins on degradation of their target proteins. Because 14-3-3s are found in organisms ranging from animals, plants and yeast, our studies could have implications into how they interact in many other processes and signaling pathways.
Intracellular Localization of Sucrose synthase
Sucrose synthase (SUS) is recognized as an important enzyme of sucrose metabolism often linked with biosynthetic processes such as cell wall synthesis and accumulation of storage products (starch, protein, oil) in developing seeds. SUS is a soluble, globular protein that has been assumed to be freely diffusing in the cytoplasm. However, recent results suggest that the enzyme may be compartmented within the cell as a result of association of SUS with membranes and F-actin in vitro in order to identify binding domains that are involved and how the interactions may be regulated. SUS has been shown to be phosphorylated on a serine residue near the N-terminus (Ser15 in maize SUS1), which may control membrane binding at least in some cases. We are speculating that the SUS at different locations within the cell may function to channel carbon into different metabolic pathways. If this is true, then controlling the localization of the enzyme may provide new strategies to control how growing cells use sucrose. These studies may also shed new light on possible microcompartmentation of other metabolic pathways. We have recently identified Ser170 as a second phosphorylation site that may target the enzyme for degradation via the ubiquitin/proteasome pathway. Understanding what distinguishes a phosphorylation site that triggers degradation from sites that do not, may provide strategies to manipulate the stability of various cellular proteins in vivo, including metabolic enzymes such as sucrose synthase.
Regulation of soybean seed composition
Developing soybean seeds receive sucrose and amino acids via the translocation steam and use these assimilates to form protein and oil (major storage products). Cultivars differ in protein:oil formation and in general, the two products are inversely related. The machanisms that control seed composition are largely unknown, but are extremely important agronomically. We are speculating that the metabolic priority for a developing seed is to utilize available amino acids for protein synthesis, and that surplus C-skeletons (in excess of that required for protein synthesis), are then available for oil production. We are postulating that this coordination is achieved at the transcriptional level, where N-metabolites regulate expression of genes encoding enzymes of starch and lipid biosynthesis; and at the post-translational level, perhaps involving protein phosphorylation of key metabolic enzymes. We are currently testing these postulates.
Protein kinase specificity
We are interested in the factors that control protein kinase specificity in plants, with particular interest in the calcium-dependent prtein kinases (CDPKs) and SNF1-related protein kinases (SnRK1s). We have identified phosphorylation motifs targeted by these kinases that may be useful in predicting new phosphorylation sites in proteins, and in application of molecular genetic approaches to modify phosphorylation-dependent processes. Fundamental studies of the biological mechanisms that control important plant prcesses may ultimately produce new approaches to increase the capacity of crop plants to produce and utilize nutrients that support growth of seeds, tubers and fruits.