Figure 2. Effects of MG132 on root-meristem size. Roots treated with 0 (a), 20 mM MG132 (b), 40 mM MG132 (c) or 80 mM MG132 (d) were stained with FM 4–64, and longitudinal views were obtained using a Zeiss confocal. The results show reduced root-meristem size (indicated by arrows) and accelerated cell differentiation (indicated by stars) in response to MG132 treatment. Bars = 20 mm. doi:10.1371/journal.pone.0045673.g002 root meristem size could also be caused by reduce of cell-division potential, we analyzed the turnover of cyclin B, an essential protein expressed during the G2 phase of the cell cycle, and degraded during mitosis [22,23]. Confocal microscopic analysis revealed that untreated Arabidopsis showed some GFP-fusion cyclin B spotted in their roots (Fig. 3 a), showing cells in M phase [24]. In contrast, both the percentage of GFP-labeled cells and the relative intensity of fluorescent signal slightly increased in roots treated with 20 mM MG132 (Fig. 3 b). And far more cyclin B signals were detected in roots treated with higher concentration of MG132 (Fig. 3 c–d). These data suggested that any level of proteasome malfunction would disturb the degradation of cyclin B, resulting in the abnormal mitosis in root meristem. Proteasome malfunction promotes, or prevents root cell elongation The final size of an organ depends on the number and volume of its cells [25]. Since proteasome malfunction disturbed cell division in the meristem zone, we therefore speculated that different concentration of proteasome inhibitors might have various effects on the cell elongation. To validate this possibility, analysis on the length of cortex cell in the maturation zone was performed. Microscopic evaluation revealed that the average cell length of untreated seedlings was about 116621 mm (Fig. 4 a). By contrast, the data of 20 mM MG132-treated seedlings was about 187623 mm (Fig. 4 b), which was approximately 61.9% longer when compared with untreated control. Though the average cell length of 40 mM MG132-treated seedlings was 143644 mm (Fig. 4 c), approximately 23.9% longer than the data of control, it was obviously shorter compared with 20 mM MG132-treated ones. On the other hand, the data of seedlings treated with 80 mM MG132 was only 87631 mm (Fig. 4 d), approximately 24.6% shorter than untreated control. These data indicated that MG132 might promote, or prevent cell elongation, depending on the level of proteasome malfunction. Roles of the UPP during Root Development Figure 3. GFP-fusion cyclin B analysis. Arabidopsis carrying GFP- fusion cyclin B were treated with 0 (a), 20 mM (b), 40 mM (c), or 80 mM (d) MG132, respectively. Longitudinal views of the GFP (left) and FM 4– 64 (middle) fluorescent emissions were collected separately and then merged (right), using a Zeiss confocal. Figures show accumulation of cyclin B in root-meristemic zone in response to MG132 treatment. Bar = 20 mm. doi:10.1371/journal.pone.0045673.g003 Figure 4. MG132 alters cell length in the maturation zone. Roots treated with 0 (a), 20 mM MG132 (b), 40 mM MG132 (c) or 80 mM MG132 (d) were stained with FM 4–64, and longitudinal views were obtained using a Zeiss confocal. The results show that MG132 alters cell length in the maturation zone (indicated by arrows). Bars = 20 mm. doi:10.1371/journal.pone.0045673.g004 PLOS ONE | www.plosone.org 4 September 2012 | Volume 7 | Issue 9 | e45673