A biochemical characterization of ubiquitin (Ub)-proteasome pathway activity was conducted in Zinnia mesophyll cell cultures to examine potential differences between differentiating cells of tracheary element (TE) cultures and non-differentiating cells of control cultures. The pathway is highly active throughout development of differentiating TEs, a programmed cell death (PCD) process during which the majority of cellular proteins and biochemical processes are expected to be down-regulated in activity and/or expression. Addition of the proteasome inhibitors clasto-lactacystin Beta-lactone (LAC) and carbobenzoxy-leucinyl-leucinyl-leucinal (LLL) at culture initiation prevented TE differentiation in this system. Proteasome inhibition at 48h did not alter the final percentage of TEs compared to controls. However, proteasome inhibition at 48 h delayed the differentiation program by approximately 24 h, as indicated by examination of morphological markers and the expression of putative autolytic cysteine proteases.These results suggest that proteasome activity is required both for induction of TE differentiation and for progression of the TE program in committed cells. Treatment at 48 h with LLL resulted in partial uncoupling of autolysis from differentiation. Results of protease activity gel analysis suggest that incomplete autolysis was due to the ability of LLL to inhibit TE cysteine proteases.

A characterization of phytohormone-stimulated growth of non-differentiating cultured Zinnia cells is also presented. Differential effects on radial cell expansion versus cell elongation were observed for the four plant growth regulators examined. Auxin (naphthaleneacetic acid, NAA) and a brassinosteroid (2,4-epibrassinolide, BI) stimulate only cell elongation. Cytokinin (N-6-benzyladenine, BA) has a greater effect on growth in cell girth rather than length. Gibberellic acid (GA₃) has equivalent effects on expansion in both dimensions. These results demonstrate that radial cell expansion and cell elongation can be uncoupled, and therefore, may be controlled by different mechanisms. Additionally, this study establishes the utility of Zinnia suspension cultures as a valuable model for studies of cell expansion.

Finally, we modified Arabidopsis plant growth conditions to promote proliferation of secondary tissues, permitting the separation of secondary xylem from bark (phloem plus nonvascular) tissues using hypocotyl-root segments. Dissected tissues were used for semi-quantitative and quantitative RT-PCR and for the construction of bark and xylem cDNA libraries for PCR-based screening of several Ub pathway components, including Ub-conjugating enzymes (UBCs), deubiquitinating enzymes (DUBs), and an Alpha (PAF1) and Beta (PAF1) subunit of the proteasome. All targeted UBC families, candidate UBCs and DUBs, and proteasome subunits are expressed in secondary xylem and bark in this system.