Figure 7. MDC staining in root cells. Seedlings treated with 0 (a), 20 mM (b), 40 mM (c), or 80 mM (d) MG132 were stained with MDC (Blue) and FM 4–64 (Red) and examined with a Zeiss confocal. The data show the accumulation of MDC in root cells in response to MG132 treatment. Bar = 20 mm. doi:10.1371/journal.pone.0045673.g007 proteasome as well as calpains [16,19]. The facts that both MG132 and MG115 showed similarly effects on root develop- ment, and that no significant differences were observed in E-64 or DMSO-treated seedlings, enabled us to confirm that the diverse effects on root growth in response to MG132 or MG115 treatment were mainly due to the inhibition of proteasome activity. Previous reports indicated that weak loss of proteasome function displayed enlarged plant organs, including cotyledons, leaves, petals, shoots and matured seeds, [7,8,12]. On the other hand, defective in proteasome activity seemly inevitably showed shorter roots [8,9,31], with the only exception that proteasome malfunc- tion stimulated the elongation of shr roots [10]. In the present study, our data revealed that lower concentration of MG132 or MG115 markedly promoted Arabidopsis root elongation, while higher concentration of the inhibitors, on the contrary, signifi- cantly inhibited root elongation (Fig. 1). Proteasome abundance and activity vary spontaneously during plant development and in response to changes in environmental conditions [11,17,32]. So we hypothesized that fluctuations in total proteasome activity might be an important mechanism for controlling the sizes of plant organs, including roots. The rate of root growth is determined by the rate of the cell division in the meristematic zone and cell differentiation in the elongation zone [1,20,25]. The control of these processes requires a number of factors and activities to be integrated in space and time. Previous reports have indicated that several proteins known to be required for the meristematic activity are believed to be Roles of the UPP during Root Development regulated by the UPP [23,33]. Furthermore, it was reported that mutation in 26S proteasome subunit RPT 2a resulted in the loss of the QC identity, as well as the cellular organization and normal nature of the RAM in Arabidopsis [9]. In the present study, our data indicated that MG132 significantly reduced the meristem size in a dose-dependent manner (Fig. 2). Moreover, confocal analysis revealed the positive relation between the accumulation of cyclin B and MG132 treatment (Fig. 3). These data are consistent and confirm the theoretical predication that proteasome-dependent proteolysis is required to control the meristematic activity, and that any level of proteasome malfunction would result in the loss of cell division potential in the root. The final sizes of determinate organs are believed to be controlled not only by cell number and size, but also by compensation, a still largely unclear mechanism that leads to the enlargement in cell size in an organ with reduced cell number [34,35]. In fact, proteasome mutation induced compensation in matured seeds, cotyledons, leaves, and petals have been observed [7,8,12]. But compensation was not believed to occur in indeterminate organs such as root [7,34]. In the present research, our new findings revealed that low concentration of MG132 markedly promoted cell elongation, resulting in the far longer longitudinal length of cells in the maturation zone (Fig. 4), which compensated MG132-induced decline in cell number. We therefore hypothesized that compensation might be a ubiquitous phenomenon occurring in all aspects of plant organ development, including root. Also, our data revealed that higher concentration of MG132 severely inhibited cell division, comparing with slightly increase or even disruption in cell elongation (Fig. 4). These effects of MG132 enabled us to propose that MG132-induced compen- sation tends to be observed only when proteasome activity still exceeds a critical threshold. And we might further hypothesize that the critical threshold of proteasome activity might be organ specific, which has indeed been reported by a previous research that all proteasome mutants tested showed larger petals than those of the wild type, but the rosette leaves were larger only in the rpt 2a and smaller in rpn10 and rpn12a [7]. Cell enlargement is often associated with endoreplication and/ or vacuolization expansion [15]. Though the UPP defection induced endoreduplication has been widely reported [8,36], the fact that cell and organ enlargement was not correlated with increased endoreduplication [8], indicating that the general increase in cell expansion in the UPP mutants was not driven by increased polyploidization. On the other hand, proteasome malfunction inevitably induced cytosolic vacuolization in a dose- and time-dependent manner in cells of both animal and plant systems [16,37–40], though the mechanism of such phenomena is still unclear. The lysosomal/vacuole dependent autophagy is another main protein degradation system characterized by the formation of autophagic vacuoles and fusing with lysosomes/vacuole to form autophagolysosomes, where proteins are degraded [41]. In the present study, immunofluorescence analysis revealed the accumu- lation of the ubiquitinated proteins, particularly in the vacuoles, in response to proteasome malfunction (Fig. 5). Besides, fluorescent staining with LysoTracker Green and MDC showed that MG132 induced dramatic autophagy in a dose-dependent manner (Figs. 6– 7). All these data have enabled us to speculate that MG132- induced accumulation of ubiquitinated proteins might be associ- ated with autophagic vacuolization as a compensatory pathway for proteolysis, which was indeed observed in the earlier investigations on animal cells. For example, Ding et al [13] revealed that autophagy was likely activated in response to endoplasmic reticulum stress caused by accumulation of ubiquitinated proteins PLOS ONE | www.plosone.org 6 September 2012 | Volume 7 | Issue 9 | e45673