Figure 1 The process of in vitro SUMOylation analysis (A) The process diagram of in vitro SUMOylation detection (Input: the key component genes of SUMO modification and the candidate substrate protein gene were introduced into Escherichia coli for expression; Activating: E1 activates SUMO molecules to participate in the modification; Conjugating: E2 attaches the activated SUMO molecules to the substrate); (B) The experimental procedure of in vitro SUMOylation detection (Co-transformation: The plasmids for expressing key SUMOylation components were introduced into E. coli, and the positive colonies were screened on LB agar plates containing kanamycin and chloramphenicol; Preparation of competent cells: The obtained positive colonies were prepared into competent cells; Co-transformation: The constructed plasmid for substrate protein expression was introduced into the competent cells mentioned above, and positive colonies were screened on LB agar plates by kanamycin, chloramphenicol and streptomycin; Induction: Protein expression was induced by IPTG; Gel electrophoresis: After lysed and denatured, samples were separated by SDS-PAGE. Then the proteins were transferred from gel to PVDF membrane; Antibody incubation: The blocked PVDF membrane was incubated with antibody; Immunoblotting: Observation and recording of the experimental results)

Figure 2 The process of in vitro SUMOylation analysis mediated by an E3 ligase (A) The process diagram of the E3 ligase-mediated in vitro SUMOylation detection (Input: The key component genes of SUMO modification and the candidate substrate protein gene were introduced into Escherichia coli for expression; Activating: E1 activates SUMO molecules to participate in the modification; Conjugating: E2 attaches the activated SUMO molecules to the substrate. In the presence of E3, E3 facilitates the attachment of SUMO molecules from E2 to the substrate protein); (B) The experimental procedure of the E3 ligase-mediated in vitro SUMOylation detection (Co-transformation: The plasmids for expression of key SUMOylation components were introduced into E. coli, and the positive colonies were screened on LB agar plates containing kanamycin and chloramphenicol; Preparation of competent cells: The obtained positive colonies were transformed into competent cells; Co-transformation: The constructed plasmid for substrate protein expression (with independent E3 expression cassette) was introduced into the competent cells mentioned above, and the positive colonies were screened by LB agar plates containing kanamycin, chloramphenicol and streptomycin; Induction: Protein expression was induced by IPTG; Immunoblotting: After lysed and denatured, samples were separated by SDS-PAGE gel electrophoresis, then the proteins were transferred from gel to PVDF membrane, the blocked PVDF membrane was incubated with antibody, the result was observed and recorded by immunoblotting).

Figure 3 Experimental cases (A) SUMOylation test of AtDREB2A; (B) Temperature affects SUMOylation of AtACR4; (C) The E3 ligase AtSIZ1 facilitates SUMOylation of AtPHR1; (D) The E3 ligase AtSIZ1 facilitates SUMOylation of AtMYB30