Different Degree in Proteasome Malfunction Has Various Effects on Root Growth Possibly through Preventing Cell Division and Promoting Autophagic Vacuolization Xianyong Sheng1,2*, Qian Wei1., Liping Jiang1., Xue Li1, Yuan Gao1, Li Wang3 1 College of Life Sciences, Capital Normal University, Beijing, China, 2 Key Laboratory of Molecular Physiology, Institute of Botany, The Chinese Academy of Sciences, Beijing, China, 3 School of Biological Science and Technology, Central South University, Changsha, China Abstract The ubiquitin/proteasome pathway plays a vital role in plant development. But the effects of proteasome malfunction on root growth, and the mechanism underlying this involvement remains unclear. In the present study, the effects of proteasome inhibitors on Arabidopsis root growth were studied through the analysis of the root length, and meristem size and cell length in maturation zone using FM4–64, and cell-division potential using GFP fusion cyclin B, and accumulation of ubiquitinated proteins using immunofluorescence labeling, and autophagy activity using LysoTracker and MDC. The results indicated that lower concentration of proteasome inhibitors promoted root growth, whereas higher concentration of inhibitors had the opposite effects. The accumulation of cyclin B was linked to MG132-induced decline in meristem size, indicating that proteasome malfunction prevented cell division. Besides, MG132-induced accumulation of the ubiquitinated proteins was associated with the increasing fluorescence signal of LysoTracker and MDC in the elongation zone, revealing a link between the activation of autophagy and proteasome malfunction. These results suggest that weak proteasome malfunction activates moderate autophagy and promotes cell elongation, which compensates the inhibitor-induced reduction of cell division, resulting in long roots. Whereas strong proteasome malfunction induces severe autophagy and disturbs cell elongation, resulting in short roots. Citation: Sheng X, Wei Q, Jiang L, Li X, Gao Y, et al. (2012) Different Degree in Proteasome Malfunction Has Various Effects on Root Growth Possibly through Preventing Cell Division and Promoting Autophagic Vacuolization. PLoS ONE 7(9): e45673. doi:10.1371/journal.pone.0045673 Editor: John Schiefelbein, University of Michigan, United States of America Received June 21, 2012; Accepted August 20, 2012; Published September 21, 2012 Copyright: 2012 Sheng et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Natural Science Foundation of China (Grant No. 30700039; 30800088) and Scientific Research Project of Beijing Educational Committee (Grant No. KM201210028017). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: xianyong.sheng@163.com . These authors contributed equally to this work. Introduction Root is a very important organ growing downward into the soil to anchor the plant as well as take up water and mineral ions. Root growth is determined by the meristem cell division and subsequently cell elongation-differentiation [1]. The control of this type of growth requires a number of factors and activities to be integrated in space and time. The ubiquitin/proteasome pathway (UPP) is one of the most important proteolytic pathways in eukaryotic cells involving in the degradation of the bulk of intracellular proteins, including misfolded proteins and short- and long-lived regulatory proteins [2–4]. It was reported that seed formation and germination was accompanied by the dynamic changes of the ubiquitinated proteins in root [5]. Besides, the mass distribution of both ubiquitinated proteins and proteasome were also observed in the root and shoot apical meristems [6]. All these data indicated that UPP was playing a role in regulating the root growth. However, the data available at present appears insufficient to provide complete knowledge of the functions of the UPP during root development. For example, analysis of several proteasome mutations with different degree in proteasome malfunction indicated that the majority of the UPP mutants have shorter root [7–9]. On the other hand, a more recent study indicated that down-regulation of the UPP activity by application of proteasome inhibitor stimulated the shr root elongation [10]. Obviously, the effects of proteasome malfunction on the root growth during postgermination development are still a debatable question. Furthermore, several researches indicated that slight loss of proteasome function leaded to an enlargement of cell size in leaves, stems, flowers, fruits, seeds, and embryos [7,8,11,12]. But so far we know very little about the mechanism underlying this phenomenon. Particularly, no attention has been paid to the possible activation of autophagic vacuolization in response to proteasome malfunction [13,14], which are closely linked to cell enlargement [15]. To extend our knowledge of the involvement of the UPP in root growth, we provide here several lines of evidence about effects of the peptide aldehyde proteasome inhibitor on Arabidopsis root growth, providing further insights into the mechanism by which the UPP controls plant growth. PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e45673