Molecular enzymology of octadecanoid signal compound biosynthesis in Arabidopsis thaliana
Final Report Abstract
In the project entitled "Molecular enzymology of octadecanoid signal compound biosynthesis in Arabidopsis thaliana'' we intended to provide detailed insight into the physical interaction between allene oxide synthase (AOS, At5g42650) and allene oxide cyclase 2 (AOC2, At3g25770) during JA synthesis. Within the project term we successfully accomplished most of the previously suggested objectives, although one final set of experiments is still pending. Executing an extended project work plan, including LOX2 in the analyses, we were able to provide conclusive evidence of a physical interaction between LOX2, AOS, and AOC2. In the first set of experiments, we were able to demonstrate that the three target proteins are capable of forming bigger complexes. In addition, we were able to illustrate that this interaction, and thereby the formation of an intimately controlled metabolic channel, is depending on the presence of membranes. In order to confirm and substantiate this finding, we established a second independent methodology in our lab. Taking into account the dependency of the target proteins on membranes to form complexes, we setup an alternative yeast two-hybrid system specially dedicated to the analysis of protein-protein interactions of membrane-bound interaction partners. Overall, this second dataset underlined the previous results, again providing evidence for the interaction of LOX2, AOS, and A0C2. As a third independent measure, we tried to directly visualize protein-protein interactions in vivo, employing bimolecular fluorescence complementation. To do so, we followed a systematic approach in which we tested the interaction of the possible partners one by one. This series of experiments, however, did not generate data that allowed drawing conclusions on the interactions between the three target proteins. This does not at all rule out the existence of such interactions, as various parameters affect the reconstitution of a functional YFP fluorophore. For example, due to folding properties of the recombinant protein, the tagcarrying terminus may be buried inside the protein. Such structural aspect would substantially impair interaction of the two YFP moieties, but not necessarily affect interaction of the target protein with its partners. Nonetheless, through these experiments we disclosed the possibility of heterocomplex formation of the various AOC isoforms in vivo. With respect to distinct overlapping expression pattern of the AOC isoforms throughout plant development, we may infer that AOC heterocomplexes play a so far underestimated role in the decision on which substrate is preferentially converted. However, it is yet too early to present a profound hypothesis, as much more experiments will be needed to explore the endogenous occurrence and ftinction of such AOC heterocomplexes. Ultimately, we tried to purify native AOS-containing protein complexes from transgenic plants. In this task the project suffered a serious setback, as the first trail to generate appropriate transgenic lines failed, which apparently perturbed the momentum of the project. Meanwhile, we have reason to speculate that we have now appropriate lines at hand. In a second attempt, we complemented the aos knockout mutant with the generated AOS-TAP-tag construct. Emphasizing the only preliminary quality of data available, we currently have four lines at hand, characterized by partial or full restoration of the wild type phenotype. In summary, this points to functional complementation of the mutation and, thus, expression and activity of the recombinant AOS introduced. However, up to date we were not able to analyze these four lines in close detail, due to the very low amount of seeds that we obtained from the T2 plants. After seed amplification, pending experiments are currently on their way. Besides all negative aspects, the delay has also a positive side, since we are now aware of the fact that the presence of membranes is essential lo stabilize the complex formed by the target proteins. Hence, we can modify and adapt the original TAP-tag purification protocol to the given circumstances. Spoken in practical terms, we have to stabilize protein interaction by chemical crosslinking before solubilization of the membrane fraction to keep the interaction partners attached to each other. So far, it is not known how the membrane can serve as a scaffold for complex formation, in particular because none of the target proteins contains a detectable transmembrane domain. At present, we cannot conclusively rule out the participation of special scaffold proteins that may help to coordinate the complex topology. The in vitro-import and crosslinking studies, however, did not provide striking evidence for the participation of such structural proteins. But there are several weakly labeled bands abundant that, taking a given stoichiometry of one scaffold protein per complex into account, may serve as potential targets for future studies though.
