Proteasome inhibitors prevent tracheary element differentiation in zinnia mesophyll cell cultures

Abstract

To determine whether proteasome activity is required for tracheary element (TE) differentiation, the proteasome inhibitors clasto-lactacystin beta-lactone and carbobenzoxy-leucinyl-leucinyl-leucinal (LLL) were used in a zinnia (Zinnia elegans) mesophyll cell culture system. The addition of proteasome inhibitors at the time of culture initiation prevented differentiation otherwise detectable at 96 h. Inhibition of the proteasome at 48 h, after cellular commitment to differentiation, did not alter the final percentage of TEs compared with controls. However, proteasome inhibition at 48 h delayed the differentiation process by approximately 24 h, as indicated by examination of both morphological markers and the expression of putative autolytic proteases. These results indicate that proteasome function is required both for induction of TE differentiation and for progression of the TE program in committed cells. Treatment at 48 h with LLL but not clasto-lactacystin beta-lactone resulted in partial uncoupling of autolysis from differentiation. Results from gel analysis of protease activity suggested that the observed incomplete autolysis was due to the ability of LLL to inhibit TE cysteine proteases.

Figure 1

Effects of inhibitor dosage and time of addition on TE differentiation. Shown are the percentages of TEs in total cells (live, undifferentiated cells plus TEs; □) detectable at 96 h following treatment with the indicated concentrations of LAC (A) or LLL (B) at t₀. Also shown are the percentages of TE detectable at 96 h following removal of inhibitors at 24 h of culture (•). C, Percentages of TEs in total cells detectable at 96 h following addition of 4 μm LAC to TE cultures at the times indicated. Values are the mean percentages ± sd of a minimum of four replicates.

Figure 2

Characterization of LLL-induced, incomplete TE autolysis. For all panels, samples were taken from TE cultures at 96 h for staining and microscopy. C and D show the same field of view. A, Bright-field microscopy of unstained control culture TEs after autolysis. B, Bright-field microscopy of unstained TEs from the t₄₈ LLL-treated culture. The two forms of protoplasmic retention by dead TEs are indicated: condensed to a single or few locations (arrowhead) or distributed throughout the cell (arrow). C, Bright-field microscopy of paraformaldehyde-fixed cells from the t₄₈ LLL-treated culture. Arrowhead indicates protoplasm retained by dead TE that failed to complete autolysis. Live TEs showed diffuse protoplasm similar to that of live, undifferentiated cells. D, Fluorescence microscopy of cells from the t₄₈ LLL-treated culture following DNA staining with Hoechst 33342. Blue fluorescence indicates the presence of nDNA; red fluorescence is due to chlorophyll autofluorescence. LT, live TE; DT, dead TE; u, live, undifferentiated cell; d, dead, undifferentiated cell (likely killed during isolation of mesophyll cells from leaves). Bar = 10 μm.

Figure 3

LAC or LLL treatment at 48 h alters the activity of TE Cys proteases. LAC (A) or LLL (B) was applied to TE cultures at t₄₈, and cells were harvested at 72 and 96 h. Shown are activity gels prepared following SDS-PAGE of total protein extracted from 1.5 × 10⁵ zinnia cells per lane (see Methods). Bands represent regions of proteolytic activity in gelatin-impregnated substrate gels. Molecular masses of protein standards are indicated on the left (in kD).

Figure 4

LLL and LAC treatments at 48 h result in elevated levels of p48h-17 expression. LLL (A) or LAC (B) was added to t₄₈ TE cultures, and cells were harvested at 72 h. Shown are RNA gel blots of total RNA performed on both an equal-cell-number (10⁶) and an equal-RNA (3.5 μg per lane) basis probed with biotinylated antisense p48h-17. RNA levels loaded for equal-cell-number analyses are as follows: LLL, 13.8 μg, and corresponding DMSO, 4.1 μg; LAC, 8.1 μg, and corresponding DMSO, 4.8 μg. Corresponding ethidium bromide-stained agarose gels are shown below each blot.

Figure 5

Endogenous ubiquitin-protein conjugates are stabilized by LLL and LAC treatments at 48 h. LLL (A) or LAC (B) was added to t₄₈ TE cultures, cells were harvested at 72 h (LLL and LAC) and 96 h (LAC only), and protein was extracted for immunoblot analysis as for Figure 3. Extracts from 1.5 × 10⁵ cells (LAC) and 2 × 10⁵ cells (LLL) were loaded per lane. Blots were probed with anti-ubiquitin antibody. Molecular masses of protein standards and the position of free ubiquitin (Ub) are indicated on the right (in kD).

Figure 6

LLL but not LAC inhibits the activity of TE Cys proteases. Following SDS-PAGE of total zinnia protein, excised gel lanes were incubated in 4 μm LAC, 20 μm LLL, or 0.1% DMSO prior to exposure to gelatin-impregnated substrate gels. Bands represent regions of proteolytic activity in substrate gels. Molecular masses of protein standards are indicated on the left (in kD).