Materials and Methods Chemicals MG132, MG115, E-64, and Dansylcadaverine (MDC) were purchased from Sigma. LysoTracker Green and FM4–64 were purchased from Invitrogen. The stock solutions of MG132 (40 mM), MG115 (40 mM), FM4–64 (500 mg ml21) and Lyso- Tracker Green (1 mM) were prepared using dimethylsulfoxide (DMSO) as solvent. E-64 (20 mM) and MDC (25 mM) were dissolved in water. Growth conditions Surface sterilized seeds of Arabidopsis thaliana (Col-0) were cold- pretreated at 4uC for 48 h, and cultured in 1/2 MS with 1% Suc for 24 h, and then uniform seedlings of similar size and primary root length were transferred to the same fresh medium containing different proteasome inhibitors MG132, MG115, and Cys- protease inhibitor E-64, for additional 48 h. Each proteasome inhibitors were used at concentrations of 0, 20, 40, and 80 mM. DMSO controls were set up by adding the similar amount of DMSO solvent. Measurement of root length Images of seedlings were acquired with a Zeiss Stemi SV11 stereoscopic microscope (Germany), equipped with an AxioCam HRc digital camera, and the lengths of the primary roots were measured using AxioVision Rel 4.8. The experiment was repeated thrice with consistent results. The values were expressed as the means 6 SD for 15 seedlings, of each treatment. FM4–64 staining Roots of both control and treated were stained with FM4–64 (2.5 mg ml21) for 2 min, and then visualized using 406 objective of a Zeiss 5 live confocal microscope equipped with an automated microscope stage and a 561 nm laser (Germany). Images were recorded and analyzed with the Zeiss Zen2007 software. Immunocytochemical analysis of ubiquitinated proteins Samples for immunocytochemical analysis were prepared as similar to earlier works [16,17]. Briefly, samples were fixed and embedded in LR White resin (Sigma). Semi-thin sections (1 mm) were blocked with 3% BSA, and then incubated with an anti- ubiquitin antibody (1:8; Sigma) and then followed by FITC- conjugated IgG (1:10; Boisynthesis, Beijing, China). All the samples were examined under the ZEISS laser scanning confocal microscope equipped with a 488 nm laser (Germany). All the sections were incubated simultaneously and using the similar antibodies, except for the negative controls that were treated as described, but omitting the primary antibody. Laser power and channel setting for FITC were kept identical for all samples to make the results comparable. Localization of cyclin B Arabidopsis with GFP-fusion cyclin B (kindly provided by associated Prof. Bai from CNU) was treated with various concentrations of MG 132. All seedlings were stained with FM 4–64 (2.5 mg ml21), and then examined under the ZEISS 5 live laser scanning confocal microscope (Germany), with a 488 nm laser for GFP, and a 561 nm laser for FM 4–64. To make the results comparable, laser power and channel setting for GFP were kept identical for all samples. Roles of the UPP during Root Development LysoTracker or MDC staining LysoTracker or MDC staining were performed according to earlier studies [18], with some modification. Roots (both control and MG132-treated) were initially incubated with LysoTracker Green (2 mM) for 25 min, or with MDC (125 mM) for 2 min. They were then washed with 1/2 MS,and incubated with FM4–64 (2.5 mg ml21) for 2 min. All samples were visualized under the confocal microscope, with a 405 nm laser for MDC, with a 488 nm laser for LysoTracker –Green, and with a 561 nm laser for FM 4–64. To make the results comparable, laser power and channel setting for MDC and LysoTracker-Green were kept identical for all samples. Statistics One-way ANOVA was used to compare the difference between the treated and control pollen tubes. Values of P,0.05 were taken as statistically significant. Results Proteasome malfunction has various effects on primary root growth To assess the roles of the UPP during root growth, seeds of Arabidopsis were cultured under standard conditions for 24 h, and then seedlings with homologous root length were treated with 0, 20, 40 or 80 mM MG132 for additional 48 h, respectively. Microscopic analysis revealed that MG132 treatment has various effects on Arabidopsis root growth in a dose-dependent manner. The seedlings cultured under control conditions for 72 h showed normal morphology with an average length of 40116785 mm (Fig. 1 a, e). In contrast, the data of seedlings treated with concentrations of MG132 were 56296764 mm (20 mM), 42336766 mm (40 mM), or 27316476 mm (80 mM) (Fig. 1 b–e). In other words, the root length of seedling treated with MG132 was approximately 40.3% longer (20 mM), or 31.9% shorter (80 mM), when compared with untreated control (P,0.05). Similar phenomena were observed when MG115, another tripeptide aldehyde, was used (Fig. 1 f, data not shown). Given that tripeptide aldehydes can also inhibit calpains as well as proteasome [16,19], the effects of E-64 as a Cys-protease inhibitor, were also investigated in our experiments. The results indicated that though 80 mM E-64 slightly inhibited root elongation, no significant differences were observed in root lengths between control and low concentration of E-64 treatment (Fig. 1 g, data not shown), indicating that effects of both MG132 and MG115 on root growth were not due to the inhibition of other protease activities. Besides, since both MG 132 and MG 115 were dissolved in DMSO, part of samples was treated with same concentration of DMSO without tripeptide aldehyde. The obtained results indicated that 0.1–0.2%, but not 0.05%, DMSO slightly promoted root growth (Fig. 1 h, data not shown), thus excluding the possible effects of DMSO on root growth. MG132 reduces meristem size through declined cell division and accelerated cell differentiation Root growth is sustained by the root meristem [20]. To clarify the mechanism by which proteasome controls root growth, we analyzed the effects of MG132 on meristem size by labeling whole- mount seedlings with FM4–64 [21]. The results indicated that the average meristem cell number of cortex cells in a file extending from the quiescent center to the first elongated cell [1], in 3-d untreated seedlings was about 3262, with a total length of about 281615 mm (Fig. 2 a). By contrast, the cell number and length of PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e45673