Chinese Bulletin of Botany ›› 2020, Vol. 55 ›› Issue (1): 83-89.doi: 10.11983/CBB19213

• TECHNIQUES AND METHODS • Previous Articles     Next Articles

Detection of SUMOylation in Plants

Qu Gaoping(),Jin Jingbo   

  1. Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
  • Received:2019-10-30 Accepted:2019-12-11 Online:2019-12-20 Published:2020-01-01
  • Contact: Qu Gaoping E-mail:qu20140701@ibcas.ac.cn

Abstract:

SUMOylation, a post-translational modification, is essential for plant normal growth and development. To date, thousands of possible SUMO substrates have been identified, but due to the relatively low SUMOylation level, biological significance of the SUMOylation remains largely unknown. Here, we summarized the SUMOylation detection methods, including in vitro and in vivo SUMOylation assays, which help to understand the functions of SUMOylation in plants.

Key words: Arabidopsis, SUMO, SUMOylation, detection methods

Table 1

Expression of SUMO reaction related proteins"

Proteins IPTG concentration
(mmol·L-1)
Induction time
His-SUMO E1 1 16-20 h (16°C)
His-SUMO E2 1 3-5 h (28°C)
His-SUMO1GG 1 16-20 h (16°C)
His-SUMO1AA 1 16-20 h (16°C)
GST-protein X-Myc 1 3-5 h (28°C)

Figure 1

Protein X can be SUMOylated in vitro GST-protein X-Myc was incubated with His-SUMO E1, His- SUMO E2 and His-SUMO1GG (His-SUMO1AA was used as a negative control) at 30°C for 3 h. After reaction, GST-protein X-Myc was purified with Glutathione beads and detected with anti-Myc and anti-SUMO1 antibodies. Arrows represent SUMOylated GST-protein X-Myc."

Figure 2

SUMOylation of GFP-protein X in Nicotiana benthamiana GFP-protein X was transiently co-expressed with FLAG-SUMO1GG or FLAG-SUMO1AA in Nicotiana benthamiana leaves. GFP-protein X was immunoprecipitated with anti-GFP antibody, and IP products were detected with anti-FLAG antibody."

Figure 3

SUMOylation of Myc-protein X in transgenic plants Myc-protein X transgenic plants and wild-type (WT) were grown in control (C) or treatment (T) conditions for 3 days. Myc-protein X and SUMOylated Myc-protein X were detected with anti-protein X and anti-SUMO1 antibodies, respectively. Vertical line indicates SUMOylated Myc-protein X bands."

Figure 4

K1 and K2 are the primary SUMOylation sites of protein X Myc-protein X, Myc-protein XK1R, Myc-protein XK2R or Myc- protein X2KR was transiently co-expressed with FLAG-SUMO1GG or FLAG-SUMO1AA in Nicotiana benthamiana leaves, respectively."

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