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Biochemical Insights on Degradation of Arabidopsis DELLA Proteins Gained From a Cell-Free Assay System^[W]

^a National Institute of Biological Sciences, Zhongguancun Life Science Park, Beijing 102206, China
^b College of Life Sciences, Beijing Normal University, Beijing 100875, China
^c Peking-Yale Joint Center for Plant Molecular Genetics and Agro-Biotechnology, National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, China
^d Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520-8104
^e State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, People's Republic of China

¹ Address correspondence to xingwang.deng{at}yale.edu.

The phytohormone gibberellic acid (GA) regulates diverse aspects of plant growth and development. GA responses are triggered by the degradation of DELLA proteins, which function as repressors in GA signaling pathways. Recent studies in Arabidopsis thaliana and rice (Oryza sativa) have implied that the degradation of DELLA proteins occurred via the ubiquitin-proteasome system. Here, we developed an Arabidopsis cell-free system to recapitulate DELLA protein degradation in vitro. Using this cell-free system, we documented that Lys-29 of ubiquitin is the major site for ubiquitin chain formation to mediate DELLA protein degradation. We also confirmed the specific roles of GA receptors and multisubunit E3 ligase components in regulating DELLA protein degradation. In addition, blocking DELLA degradation with a PP1/PP2A phosphatase inhibitor in our cell-free assay suggested that degradation of DELLA proteins required protein Ser/Thr dephosphorylation activity. Furthermore, our data revealed that the LZ domain of Arabidopsis DELLA proteins is essential for both their stability and activity. Thus, our in vitro degradation system provides biochemical insights into the regulation of DELLA protein degradation. This in vitro assay system could be widely adapted for dissecting cellular signaling pathways in which regulated proteolysis is a key recurrent theme.