Microtubules (MTs) are dynamic cytoskeletal polymers of alpha-(TUA) and beta-(TUB) tubulins. Various processes including the deposition of cellulose microfibrils in the developing cell wall are thought to depend on MT function. In turn, post-translational modifications (PTMs) at the C-terminal end of the tubulin monomers contribute to the regulation of MT growth and stability. In order to investigate the importance of MTs during cell wall formation, a small subset of xylem-abundant tubulins (TUA1, TUA5, TUB9 and TUB15) including PTM mimics of TUA1 (dY and dEY) were expressed ectopically in transgenic Populus. Out of several TUA - TUB gene pairs used for transformation, only combinations containing PTM mimics of TUA1 led to transgenic plants. Plants expressing the TUA1dY+TUB9 or TUA1dEY+TUB15 combinations were used for further characterization. Transgenic plants exhibited changes in cell wall properties, although cellulose, hemicellulose and lignin contents were unaffected. Based on cell wall glycome profiling, the pectin-xylan polysaccharide matrix was altered in stem wood of both transgenic groups. Lignin composition was altered in the transgenics which exhibited decreased S/G monomer ratios. Guard cell dynamics are highly dependent upon MT dynamics, and therefore, leaf gas exchange characteristics were determined in plants exposed to short- or long-term water deficits. During a short-term, acute drought stress, source leaf transpiration and net photosynthesis rates were higher in transgenic than wild type plants. Leaf gas exchange characteristics did not differ between transgenic and wild type plants maintained at chronically reduced soil water potential. Mature leaves also exhibited greater width-to-length ratios in TUA1dY+TUB9 compared to the other plant lines. The results suggested that tubulin manipulations in Populus had pleiotropic effects on cell wall deposition, guard cell dynamics and cell expansion.