MG132-induced accumulation of ubiquitinated proteins is associated with autophagic vacuolization In the present study, we also found that MG132 treatment is concomitant with strongly cytoplasmic vacuolization in the elongation zone (Data not shown). Given that most substrate proteins are usually ubiquitinated before being degraded by the proteasome [2–4], and that inhibition of proteasome activity induces toxic accumulation of ubiquitinated proteins [16]. We therefore speculated that MG132-induced toxic accumulation of ubiquitinated proteins might be associated with the formation of cellular vacuolization in root cells. To validate this speculation, immunofluorescence analysis was carried out using anti-ubiquitin antibody. The obtained results revealed that detectable fluorescent signal was seen in both nuclei and cytoplasm in untreated root cells (Fig. 5 a). On the other hand, a far brighter fluorescence was detected in root treated with 20 mM MG132 (Fig. 5 b). Particularly, strong dot-pattern antigen signal were observed in almost every vacuoles, which were rarely observed in untreated roots (Fig. 5 a). And the increase in the intensity of fluorescence became more obvious in roots treated with 40 mM MG132 (Fig. 5 c). But the magnitude of fluorescent intensity was not significantly higher in root treated with 80 mM MG132 than in that treated with 40 mM MG132 (Fig. 5 d). Previous reports on animal system have indicated a crosstalk between the UPP and lysosome/vacuole-dependent autophagy [14,26]. We therefore further speculated that proteasome malfunction might also activate autophagy in root cells. To verify this possibility, we initially stained the untreated and MG132- treated roots with Lysotracker-Green, a fluorescent acidotropic probe widely used to visualize acidic compartments such as lysosome/vacuole [27]. The obtained results revealed that a faint fluorescent signal could be detected in untreated root, mainly in the elongation zone, but not in the meristem zone (Fig. 6 a). In contrast, LysoTracker-stained structures were obviously observed in 20 mM MG132-treated roots, in both the meristem zone and elongation zone (Fig. 6 b). And the fluorescent intensity becomes far stronger as the concentration of MG132 increases to 40 mM (Fig. 6 c). Larger LysoTracker-stained structures were usually Figure 5. Immunofluorescence labeling of ubiquitinated pro- teins in root cells. Semi-thin sections of roots treated with 0 (a), 20 mM MG132 (b), 40 mM MG132 (c) or 80 mM MG132 (d) were incubated with anti-ubiquitin antibody and FITC-IgG, and examined with a Zeiss confocal. Arrows show the MG132-induced accumulation of ubiquitinated proteins in vacuoles. Bar = 20 mm. doi:10.1371/journal.pone.0045673.g005 Roles of the UPP during Root Development Figure 6. LysoTracker staining in root cells. Seedlings treated with 0 (a), 20 mM (b), 40 mM (c), or 80 mM (d) MG132 were stained with LysoTracker (Green) and FM 4–64 (Red), and examined with a Zeiss confocal. The data show the accumulation of LysoTracker in root cells in response to MG132 treatment. Bar = 50 mm. doi:10.1371/journal.pone.0045673.g006 observed in 80 mM MG132-treated roots, the magnitude of fluorescent intensity was not significantly higher in 80 mM MG132- treated roots than that in 40 mM MG132-treated ones (Fig. 6 d). Though the ability of LysoTracker to label autophagy in plants has been demonstrated in various organisms including Arabidopsis [18,28,29], it was believed that the dye must be used in combination with other markers of autophagy in order to discriminate autophagic activity from other events increasing lysosome/vacuole activity [27]. We thus used MDC, a commonly used autophagic dye, as a more specific marker to detect autophagy [27,30]. The results indicated that few MDC-stained structures were visible in the elongation zone of untreated root (Fig. 7 a). In contrast, 20 mM MG132-treated cells contained numerous cytoplasmic structures that concentrated MDC (Fig. 7 b) that were similar in appearance to the starvation-induced autolysosomes in BY-2 cells [30]. And far more MDC-stained structures were observed in 40 mM MG132 treated roots (Fig. 7 c). Strikingly, the amounts of MDC-stained structures seemed to be the highest in 80 mM MG132-treated roots (Fig. 7 d). These data demonstrated the dose-dependent activation of autophagy in response to proteasome malfunction in roots. Discussion Application of proteasome inhibitors is one of the main strategies to study the role of the UPP during plant development. In the present study, we utilized MG132 and MG115, two widely used proteasome inhibitors in both plant and animal systems, to determine the effects of proteasome malfunction on primary root development. The obtained results indicated that different concentration of MG132 or MG115 had diverse effects on root growth (Fig. 1). Nevertheless, the peptide aldehyde may inhibit PLOS ONE | www.plosone.org 5 September 2012 | Volume 7 | Issue 9 | e45673