Three distinct K$ sp+$ currents were identified in corn (Zea mays) protoplasts using the whole-cell patch-clamp technique. Inward-rectifying K$ sp+$ currents were evoked at membrane potentials more negative than $-$100 mV. The activation range was sensitive to external K$ sp+$ and shifted in the positive direction as the K$ sp+$ concentration was elevated. The second K$ sp+$ current was voltage-independent and contributed to the resting membrane conductance of the protoplast. Finally, a voltage- and Ca$ sp{2+}$-dependent K$ sp+$ current was observed at potentials positive to $-$60 mV. This current was inhibited by reagents which antagonize plasmalemma Ca$ sp{2+}$ influx (e.g. nitrendipine, verapamil). In contrast, currents were enhanced by increasing the cytosolic free Ca$ sp{2+}$ concentration from 40 to 400 nM. The Ca$ sp{2+}$-dependent K$ sp+$ current was inhibited by tetraethylammonium ions, Cs$ sp+$, Ba$ sp{2+}$, and charybdotoxin which suggested that the channel protein has structural similarities to the high conductance Ca$ sp{2+}$-dependent K$ sp+$ channel observed in animal systems.

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Stomata are specialized plant structures required for gaseous exchange with the outer environment. During stomata formation, the cytoskeleton plays an important role in controlling the division of the individual cells leading to the generation of the stomata complex. Two mutants that affect microfilament and microtubule organization in subsidiary mother cells include brk1 and dcd1. While only 20% of the subsidiary cells in the brk1 and dcd1 single mutants are abnormally shaped, it was reported that there is a synergistic effect between the brk1 and dcd1 mutations in the brk1; dcd1 double mutant since 100% of the subsidiary cells are abnormal. The focus of this research is to try to understand this synergistic effect by investigating the actin cytoskeleton and nuclear position in the single and double mutants. The reported results include the observation that the size of actin patch was largest in the wild-type subsidiary mother cells (SMCs) and smallest in dcd1 and brk1; dcd1 SMCs and that brk1 and brk1; dcd1 double mutants had fewer actin patches than wild-type and dcd1 SMCs. Additionally, we observed that some SMCs that did not have actin patches still underwent nuclear migration suggesting that nuclear migration may not be solely dependent on actin patch formation. Finally, during SMC cytokinesis, a large percentage of double mutant (brk1; dcd1) cells showed an off-track development of the phragmoplast as compared to the single mutants and the wild-type plant explaining the large number of abnormally shaped subsidiary cells in the double mutants.

Plant microtubules play essential roles in cell processes such as cell division, cell elongation, and organelle organization. Microtubules are arranged in highly dynamic and ordered arrays, but unlike animal cells, plant cells lack centrosomes. Therefore, microtubule nucleation and organization are governed by microtubule-associated proteins, including a microtubule-severing protein, katanin. Mutant analysis and in vitro characterization has shown that the highly conserved katanin is needed for the organization of the microtubule arrays in Arabidopsis and rice as well as in a variety of animal models. Katanin is a protein complex that is part of the AAA+ family of ATPases. Katanin is composed of two subunits, katanin-p60, a catalytic subunit and katanin-p80, a regulatory subunit. Spastin is another MT-severing protein that was identified on the basis of its homology to katanin. In animal cells, spastin is also needed for microtubule organization, but its functionality has not yet been investigated in plants. To initiate an exploration of the function of katanin-p60 and spastin in Zea mays, my research goal was to generate tools for the expression and purification of maize katanin-p60 and spastin proteins in vitro. Plasmids that express katanin-p60 and spastin with N-terminal GST tags were designed and constructed via In-Fusion® cloning after traditional cloning methods were not successful. The constructs were expressed in E. coli, then the recombinant proteins were purified. To determine if the GST-tagged proteins are functional, ATPase activity and tubulin polymerization assays were performed. While both GST-katanin-p60 and GST-spastin hydrolyzed ATP indicating that the ATPase domains are functional, the results of the tubulin polymerization assays were less clear and further experimentation is necessary.

Eighteen-day-old seedlings of five maize hybrids (A619xH60, B73xM017, B73xPA91, B73xVA17 and A632xH96) were grown hydroponically and compared for inherent differences in lipid concentration and composition. These seedlings were also evaluated for their ability to tolerate mild osmotic stresses (-0.4 and -0.6 MPa, polyethylene glycol-induced), osmotically induced changes in lipid composition, and differences in membrane stability as measured by electrolyte leakage. Inherent differences among the hybrids included reduced dry matter accumulation, and lower total lipid and free fatty acid concentration in the leaves and roots of B73xVA17 and A632xH96 compared to A619xH60, B73xMOl7, and B73xPA91. No differences were apparent in the frea, glycosidic or esterified steryl fractions among hybrids. Distributional patterns of fatty acid and sterol composition differed among tissues but were similar in all hybrids. Osmotic treatments of -0.4 and -0.6 MPa resulted in significant reductions in the dry weight of B73xVA17 and A632xH96. Total lipid concentration increased significantly in the roots of all hybrids while there was a general trend for moderate increases in stems and leaves. Few, mostly insignificant differences, were observed in the free fatty acid, free sterol, steryl glycoside and steryl ester fractions. However, the stigmasterol to sitosterol ratio increased in all three steryl fractions in the roots of B73xVA17 and A632xH96 as a result of the osmotic treatments. B73xVA17 and A632xH96 also exhibited the greatest electrolyte leakage when leaf discs were subjected to osmotic stress. B73xVA17 and A632xH96 appear to be less· tolerant to osmotic stress than the other hybrids. This may be due to their comparatively earlier developmental growth stage at the onset of osmotic stress. Modification of lipids in these sensitive hybrids may reflect an initial stabilization of cellular membranes which, in turn, may have some adaptive value in terms of drought tolerance. / M.S.