Phosphatidylinositol phosphate 5-kinases (PIPKs) are the enzymes that synthesize PtdIns(4,5)P2, an important signaling lipid found in plants and animals. Recent comparative biochemical characterizations make a compelling argument that plant PIPKs are unique, multifunctional proteins which define plant phosphoinositide (PI) signaling. In contrast to mammalian PIPKs, Arabidopsis PIPK1 (AtPIPK1) binds directly to actin without needing adaptor proteins. Furthermore, AtPIPK1 has an extended N-terminal domain that binds negatively charged lipids including PtdIns(4,5)P2. The ability to both synthesize and sequester PtdIns(4,5)P2 makes this the ultimate PIPmodulin, a protein predicted to sequester and modulate the impact of PtdIns(4,5)P2 on cellular physiology. No other organism (including mammals, yeast, C. elegans and Drosophila) has PIPKs that contain similar N-terminal motifs or that bind directly to actin. The hypothesis to be tested is that the plant-specific PIPKs connect membrane lipids to fine actin filaments and that hyperosmotic stress affects both this connection and the activity of the PIPKs. This work will reveal new insights into the mechanisms through which these plant-specific enzymes connect the actin cytoskeleton to membranes and generate PtdIns(4,5)P2-enriched microdomains.
The research will have a major impact on the understanding of the fundamental differences in PI metabolism in plants and animals. The research also will impact the careers of one postdoc, one graduate student and at least 3 undergraduates a semester who will work with the group. The undergraduates will be given independent research projects to generate and characterize the recombinant proteins and will help with transforming protoplasts. The undergraduates will write research proposals for the annual University-wide competition and will give a poster presentation on their research at the undergraduate research symposium. Undergraduates in the PIs program routinely win awards for their presentations. Moreover several young scientists from the program have entered into careers in plant biology.
The phosphoinositide signaling pathway is involved in plant responses to many abiotic and biotic stresses. In this pathway, the membrane-associated phospholipid, phosphatidylinositol (PtdIns) is sequentially phosphorylated by specific lipid kinases to form phosphatidylinositol 4-phosphate (PtdInsP) and phosphatidylinositol 4,5 bisphosphate (PtdInsP2). In response to a stimulus, PtdInsP2 is hydrolyzed by phospholipase C (PLC) to produce the soluble second messenger inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG). A major conundrum in the field is why levels of PtdInsP2 are so low in plants. We addressed this in 2 ways. First, we have biochemically characterized the plant enzymes critical for phosphoinositide signaling, the PIP kinases. We compared the enzymes from humans with the plant enzymes and found that the plant enzymes were less active in vitro and were regulated by an additional N-terminal lipid binding domain. The characterization of these enzymes is leading to new insights into the plant-specific regulation of phosphoinositide signaling. We are developing in vivo tagged proteins which will enable scientists to identify interacting protein and lipid complexes and will extend our understanding of the PI pathway from the plasma membrane to the nucleus. Second, we took a synthetic approach to identify flux limiting steps in the PI pathway and identify downstream events. Because the pathway is more robust in humans compared to plants, we expressed human genes that would either decrease or increase PI signaling in plants. Using this approach we were able to show that lipid synthesis was a flux limiting step in plants. We also identified downstream events affected by PI signaling and showed for the first time that about 30% of a stress-induced calcium signal in plants was InsP3 -mediated. In a collaborative effort, we showed that increasing the inositol lipids in the plant plasma membrane affected the potassium efflux channel activity. We have also gained insight into the importance of the phosphoinositide pathway in abiotic stress responses. Signal transduction is difficult to study in living cells because the generation of second messengers is rapid and transient. By producing plants with constitutively altered signaling, we can identify downstream pathways that are affected (up or down regulated) by the presence and equally importantly, the absence, of the signal. These plants are valuable research tools to study the role of phosphoinositide signaling in various plant responses and to identify the downstream components in the pathway. Understanding how plants sense and respond to different stresses provides valuable knowledge towards improving plant productivity under changing environmental conditions.
