The objective of this award is to open up new, biophysical, approaches to studying regulation of the plant as it undergoes development and as it experiences environmental viscissitudes. Although one of the most important sites of regulation the plant cell periphery, which interfaces with neighbor cells and with the environment has been historically difficult to study with biochemical techniques, emerging biophysical methods are permitting kinds of data acquisition and analysis that promise to raise understanding to a new level. These techniques are valuable because of the very fact that the periphery of the plant cell is a mechanical structure in which complex load-bearing elements play feedback roles with biochemical signals and pathways. The system cannot be understood without mechanical analysis. The experiments of this project combine high-resolution multidimensional microscopy of living cells with a mathematical method called finite element analysis to elucidate how one common but newly discovered set of peripheral structures helps stabilize cells mechanically under set conditions yet participates in transitions to new cell states as developmental and environmental parameters change. Particular focus will be on mechanical stresses such as wind and desiccation.
These interdisciplinary studies should suggest new tools for agricultural stress management and approaches to genetic manipulation of crop plants. More broadly, successes will direct attention to the timely effort to expand the important field of plant biophysics, historically overshadowed by biochemistry and molecular biology. Outreach plans will bring underprivileged public high school girls to Washington University for "Saturdays with a Scientist": interdisciplinary labs, lectures, historical perspectives and social interactions to help them realize possibilities offered by rigorous study of the sciences. Social interactions will be maintained with these girls throughout high school to provide role models and informal career counseling as they progress academically, develop their goals and, presumably, consider application to universities.
Project One We have analytically modeled and imaged a novel way that growing cells of the experimental plant Arabidopsis use the previously mysterious regulatory glycoprotein (or carbohydrate-bearing protein) named lysine-rich arabinogalactan protein to enable and orient cell growth. The accompanying images illustrate two specific findings. One represents some of our nano-scale visualizations of the glycoprotein itself, a "first" for the atomic force microscopy of such a plant polymer. The other represents our resolution by fluorescence microscopy of the way in which the polymers associate with each other to form "fibrils" next to the cell membrane. These fibrils are oriented in much the way as are cellulose fibrils recently deposited at the membrane, and we posit that they are helping to maintain order in this system by acting as spacers. We suggest that without such spacers between small fibrils of cellulose, the latter would clump into large fibrils rather than being maintained at the appropriate functional size to generate the cell walls. New layers of cellulose and glycoprotein are added at the membrane to thicken the cell wall during its development, but this must be orderly if pattern is to be maintained. Not only the distance between cellulose fibrils but also the orientation of the fibrils is important, for orientation has long been known to mechanically constrain the directions in which a cell can grow. One of two manuscripts describing this study has been published at the time of this report. Understanding the basic mechanisms of regulated cell growth is considered fundamental for development of agricultural technologies. Furthermore, understanding the formation of cell walls is important for improving ways to use wall products for fuel and for industrial products. Project Two We have discovered an electromechanical early warning system giving the Arabidopsis plant notice of the arrival of potentially predatory insects. This system consists of hairs called trichomes that have evolved a mechanically elaborate wall that is able to transmit and actually focus force to a basal ring of cells if the wall is poked or rubbed. The stimulated basal cells initiate elaborate oscillations of the "universal regulatory ion" calcium, and it appears that heightened synthesis of insect-deterrent toxins occurs in these cells and the neighboring layer of cells that cover the leaf. Moreover, little bumps called papillae on the surface of the trichome release accumulated deterrents if they are bent or rubbed. This system is of great agricultural and ecological interest, because it is well documented that the constitutive production and storage of toxins has a high cost such that the more insect repellant is accumulated the less material and energy a plant can direct to growth and seed production. It seems clear that this early warning system, which likely occurs in many varieties of plants, could be utilized agriculturally to diminish the financial and ecological costs of treatment with synthetic pesticides. Trichome mechanosignaling came to our attention because we were studying at high resolution the general distribution of a particular member of the arabinogalactan protein class studied in Project One, above. Based on its structure at the molecular and nanometer levels, we had already postulated that it has mechanical regulatory properties. Interestingly, this particular glycoprotein is uniquely distributed in the papillae. and we speculate that--like the more general cell types initially described--this is an excellent cell system in which we can explore the mechanism by which the glycoprotein exerts its evidently electromechanical role. There are further benefits to this trichome research. Study of the wall has revealed a novel realm in which it appears that both biochemical signaling and biosynthesis occur. Evidently, as it evolved for force focusing to control toxin synthesis, the wall also developed special compartments which, together with cytoplasmic compartments, enable separation of relatively nontoxic precursors and enzymes that act on them to produce the ultimately pesticides, so that regulated production of the toxins can occur safely outside the specially vulnerable cytoplasm under appropriate conditions. Previously, separate compartments within the cell cytoplasm itself had been identified, but with the participation of the wall finer capabilities of control are apparently enabled. Consistent with this architecture, six new kinds of compartmented signaling have been discovered in the wall. This is interesting because in former years the wall was considered inert. Understanding regulatory steps is generally an excellent means by which humans can unlock the controls evolved by the plant. Again, we expect to learn that some of these features are generally, if less dramatically, expressed in several other plant varieties, and speculate that further study of the signals within the cell wall could lead to beneficial agricultural practices. Just as the first project with the unique regulatory glycoprotein led to the second, the second has led to several new kinds of electromechanical signaling in Arabidopsis that we will continue exploring in the future.
