Across the tree of life, rotary molecular motors like the F1FO ATP synthase utilize a transmembrane nonequilibrium proton gradient to synthesize adenosine triphosphate (ATP), the biological energy currency. The catalytic portion of rotary motors, such as the F1 complex from E. coli and the V1 complex from S. cerevisiae, was purified and studied during ATP hydrolysis. Single-molecule assays utilized gold nanorods to investigate the kinetics of the F1-ATPase catalytic dwell, the biophysics of V1-ATPase, and the kinematics of the F1-ATPase power stroke. Observation of oscillatory rotor motion during the F1 catalytic dwell provided new insight as to how energy from ATP binding is stored during its three stages. That motion indicated a ratchet mechanism, in which F1 changed states according to first-order kinetics with a time constant τ = 0.182, showing that Stage-1 represents a pre-hydrolysis state and Stage-2 represents a post-hydrolysis state. F1 was then observed to return to 0° prior to its next power stroke (Stage-3), which explained why the three catalytic dwells remain 120° apart after many revolutions. Analysis of the 120° power stroke following Stage-3 was conducted in both V1 and F1, allowing comparative biology to elucidate defects in the ATPase mechanism, such as ADP inhibition and faltering rotation. It is noteworthy that the V1 rotary positions of ADP release and ATP binding are the opposite of F1, and that less elastic energy is stored in the V1 rotor due to differences in its catch loop. In both rotary ATPases, energy contributed by binding and hydrolysis can dissipate at multiple points. When the F1 catch loop contact between F1 βD305 and γQ269 was mutated, the elastic energy stored in the rotor dissipated dramatically. Dissipation was clearly shown by sustained Phase-1 decelerations, the distribution of ATP-binding dwells, and high-amplitude oscillations in γQ269L. These findings clarify evolutionary similarities and differences between eukaryotic V1, which is exclusively a hydrolase, and F1, which can both hydrolyze and synthesize ATP.

The remarkable conservation of molecular and intra-/inter-cellular pathways underpinning the fundamental aspects of sugar partitioning in two evolutionarily divergent organisms – a non-vascular moss Physcomitrella patens and a vascular cereal crop Oryza sativa (rice) – forms the basis of this manuscript. Much of our current knowledge pertaining to sugar partitioning in plants mainly comes from studies in thale cress, Arabidopsis thaliana, but how photosynthetic sugar is loaded into the phloem in a crop as important as rice is still debated. Even less is known about the mechanistic aspects of sugar movement in mosses. In plants, sugar either moves passively via intercellular channels called plasmodesmata, or through the cell wall spaces in an energy-consuming process. As such, I first investigated the structure of plasmodesmata in rice leaf minor vein using electron tomography to create as of yet unreported 3D models of these channels in both simple and branched conformations. Contrary to generally held belief, I report two different 3D morphotypes of simple plasmodesmata in rice. Furthermore, the complementary body of evidence in arabidopsis implicates plasma membrane localized Proton Pyrophosphatase (H+-PPase) in the energy-dependent movement of sugar. Within this wider purview, I studied the in situ ultrastructural localization patterns of H+-PPase orthologs in high-pressure frozen tissues of rice and physcomitrella. Were H+-PPases neo-functionalized in the vascular tissues of higher plants? Or are there evolutionarily conserved roles of this protein that transcend the phylogenetic diversity of land plants? I show that H+-PPases are distinctly expressed in the actively growing regions of both rice and physcomitrella. As expected, H+-PPases were also localized in the vascular tissues of rice. But surprisingly, H+-PPase orthologs were also prominently expressed at the gametophyte-sporophyte junction of physcomitrella. Upon immunogold labeling, H+-PPases were found to be predominantly localized at the plasma membrane of the phloem complexes of rice source leaves, and both the vacuoles and plasma membrane of the transfer cells in the physcomitrella haustorium, linking H+-PPases in active sucrose loading in both plants. As such, these findings suggest that the localization and presumably the function of H+-PPases are conserved throughout the evolutionary history of land plants.